Cellular quality control provides an efficient surveillance system to regulate mitochondrial turnover. This study elucidates a new interaction between the cytosolic E3 ligase mahogunin RING finger 1 (MGRN1) and the endoplasmic reticulum (ER) ubiquitin E3 ligase GP78 (also known as AMFR). Loss of Mgrn1 function has been implicated in late-onset spongiform neurodegeneration and congenital heart defects, among several developmental defects. Here, we show that MGRN1 ubiquitylates GP78 in trans through non-canonical K11 linkages. This helps maintain constitutively low levels of GP78 in healthy cells, in turn downregulating mitophagy. GP78, however, does not regulate MGRN1. When mitochondria are stressed, cytosolic Ca2+ increases. This leads to a reduced interaction between MGRN1 and GP78 and its compromised ubiquitylation. Chelating Ca2+ restores association between the two ligases and the in trans ubiquitylation. Catalytic inactivation of MGRN1 results in elevated levels of GP78 and a consequential increase in the initiation of mitophagy. This is important because functional depletion of MGRN1 by the membrane-associated disease-causing prion protein CtmPrP affects polyubiquitylation and degradation of GP78, also leading to an increase in mitophagy events. This suggests that MGRN1 participates in mitochondrial quality control and could contribute to neurodegeneration in a subset of CtmPrP-mediated prion diseases.

Ubiquitylation of proteins is an essential regulator of the cellular machinery and has multiple roles in maintaining homeostasis. Addition of ubiquitin molecules to cellular substrates act as signals that either result in proteasomal degradation of targets or regulate their function. The ubiquitylation process involves an E1 ubiquitin-activating enzyme, an E2-conjugating enzyme and an E3 ubiquitin ligase. The E3 ubiquitin ligase imparts substrate specificity (Hershko and Ciechanover, 1998). Ubiquitylation-mediated regulation can be complex because several E3 ligases can act together to modulate cellular processes. The regulation of degradation of the E3 ligases remains a relatively unexplored area. They can be degraded by the proteasome through two main mechanisms – self-catalyzed ubiquitylation and/or the activity of an exogenous ligase. Self-ubiquitylation, the hallmark of E3 ligases, has long been considered to target them for degradation. However, it turns out that many of them, even those that catalyze their own ubiquitylation, are targeted in trans by exogenous ligases. Similarly, self-ubiquitylation has been implicated in the regulation of their activity and need not necessarily target these proteins for degradation (Weissman et al., 2011; de Bie and Ciechanover, 2011).

Several ligases that can mediate their own degradation have also been shown to be regulated by other external ligases. One such ligase is mouse double minute (Mdm2), which can direct its own ubiquitylation and subsequent proteasomal degradation (Ranaweera and Yang, 2013). In parallel, it has also been reported that the histone acetyl transferase p300-CBP-associated factor (PCAF, also known as KAT2B) ubiquitylates Mdm2, resulting in its proteasomal degradation (Linares et al., 2007). It has been proposed that self-induced degradation of Mdm2 serves as a backup mechanism that occurs only when its level exceeds a certain threshold (Song et al., 2008). Similarly, GP78 (also known as AMFR), a RING finger ligase implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded proteins, can self-ubiquitylate leading to its own degradation (Fang et al., 2001). In addition, it is also targeted for proteasomal degradation by HRD1 and in turn affects the levels of insulin-induced gene-1 (Insig-1) (Ballar et al., 2010; Shmueli et al., 2009). GP78 might also be ubiquitylated by tripartite motif-containing protein 25 (TRIM25) for proteasomal degradation, although the physiological relevance of this is unknown (Wang et al., 2014). The E3 ligases of the CBL (named after Casitas B-lineage lymphoma) family, known to ubiquitylate and downregulate growth factor receptors, also appear to be regulated by other ligases in trans. The homologous to the E6-AP C-terminus (HECT) E3 ligases NEDD4 and ITCH mediate degradation of CBL proteins to reverse their effects on receptor downregulation and signaling (Courbard et al., 2002; Magnifico et al., 2003; Yang et al., 2008; Gruber et al., 2009).

GP78 is an E3 ligase that is linked to tumor metastasis as a receptor of autocrine motility factor. It has also been established that it mediates ubiquitylation of ERAD substrates like cystic fibrosis transmembrane conductance regulator (CFTR) and apolipoprotein B (APOB) for proteasomal degradation, thereby playing an important role in this cellular process (Liang et al., 2003; Morito et al., 2008). Recent studies further highlight a role of GP78 in mitophagy. Overexpression of functional GP78, but not its catalytic RING domain mutant, causes perinuclear mitochondrial clustering. This leads to increased ubiquitylation and degradation of mitofusins along with an increase in recruitment of LC3 (MAP1LC3A) to the mitochondria-associated ER (Fu et al., 2013). Degradation of mitofusins by GP78 is depolarization dependent, as this occurs in the presence of carbonyl cyanide m-chloro phenyl hydrazone (CCCP), thus suggesting a role for GP78 in quality control of depolarized mitochondria. Here, we show that the protein levels of GP78 are in turn controlled by mahogunin RING finger 1 (MGRN1)-mediated ubiquitylation in a CCCP-dependent manner, thereby providing a higher order of regulation of mitochondrial health.

MGRN1 is a cytosolic RING-domain-containing E3 ligase, loss of which has been implicated in mahoganoid coat color, adult-onset spongiform neurodegeneration (phenotypically similar to prion diseases), reduced embryonic viability (with 46–60% mortality of homozygotes by weaning age) and developmental defects (including heterotaxia and congenital heart defects) in mice (He et al., 2003; Cota et al., 2006; Chakrabarti and Hegde, 2009; Jiao et al., 2009). Although recent studies have suggested that MGRN1 has a role in oxidative stress (Sun et al., 2007; Chhangani and Mishra, 2013), the molecular basis for these observations was elusive.

Our study shows that MGRN1 can affect the mitochondria by modulating GP78. MGRN1 interacts with and ubiquitylates GP78 through non-canonical K11 lysine linkages, thereby targeting it for proteasomal degradation. This ubiquitylation is CCCP dependent, as it occurs in normal cells but decreases with CCCP treatment, resulting in concomitantly higher GP78 levels and favoring mitophagy of depolarized mitochondria. The presence of CCCP leads to a rise in cytosolic Ca2+, which is detrimental for the interaction of MGRN1 with GP78 and hence its ubiquitylation. BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], a chelator of Ca2+ ions can reverse these effects. This study is important because perturbation of MGRN1 function in the presence of disease-causing PrP mutants compromises polyubiquitylation of GP78, suggesting that MGRN1 participates in mitochondrial biogenesis and dysfunction in CtmPrP-mediated neurodegeneration.

Depletion of MGRN1 results in altered mitochondrial distribution

We observed in different cell lines and primary cells that the typical well spread-out pattern of mitochondrial distribution was altered upon functional depletion of MGRN1 (Fig. 1). HeLa cells treated with MGRN1 small interfering RNA (siRNAs) or transiently expressing catalytically inactive MGRN1 lacking the RING domain (MGRN1ΔR) showed perinuclear clustering of mitochondria with a reduction in the mitochondrial distribution when compared to control cells treated with mock siRNA or those expressing functional MGRN1 (Fig. 1A,B; Fig. S1A; see also Movies 1 and 2). The altered mitochondrial distribution in MGRN1-depleted cells was very similar to that observed in cells overexpressing GP78 (Fu et al., 2013) (Fig. S1B,C). To validate the role of MGRN1, siRNA-treated HeLa cells were subjected to rescue experiments. Expression of MGRN1 rescued the clustering phenotype, whereas expression of MGRN1ΔR could not (Fig. 1C,D). The alteration in mitochondrial distribution was independent of the cell line, as it was detected in HeLa cells, SHSY5Y cells and primary mouse embryonic fibroblasts (MEFs) (Fig. 1E–G). Immunostaining of cells treated with MGRN1 siRNA for cytochrome c oxidase subunit IV (COX4), and confocal imaging of live cells co-transfected with MGRN1 or MGRN1ΔR together with mitoRFP also showed that these cells had perinuclearly clustered mitochondria (Fig. S1D–F). MGRN1-null melanocytes (denoted melan md1-nc) did not have mitochondrial clusters (Fig. S1G). Altered mitochondrial distributions were also seen on treating most cell lines (such as HeLa cells) with CCCP irrespective of the MGRN1 status. Melanocytes, however, did not show this phenotype, which might account for the lack of mitochondrial clustering in MGRN1-null melanocytes (Fig. S1H).

Fig. 1.

Depletion of MGRN1 causes perinuclear clustering of mitochondria. (A) HeLa cells were treated with MGRN1 siRNA or mock siRNA or transfected with MGRN1 or MGRN1ΔR and imaged. Mitochondria were marked by mitoGFP. Note perinuclear clustering of mitochondria in cells upon the depletion of MGRN1. Images are 3D projections obtained from z-stacks using ImageJ; MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. Scale bars: 10 μm. The border of the transfected cell is marked by the dotted line. (B) The mitochondrial distribution was calculated with ImageJ for cells imaged in A using their z-projections. Graph shows mean±s.e.m. from ∼150 cells analyzed from five independent experiments. ***P≤0.001; ns, not significant (P=0.2) (unpaired two-tailed Student's t-test). (C) HeLa cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1 or MGRN1ΔR and mitoGFP after 48 h of siRNA treatment. z-stacks were taken 24 h later. Ectopic expression of MGRN1 but not MGRN1ΔR could rescue the mitochondrial clustering in MGRN1-depleted cells. MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. The border of the transfected cell is marked by the dotted line. Scale bars: 10 μm. (D) The mitochondrial distribution was calculated with ImageJ for cells imaged in C using their z-projections. Graph shows mean±s.e.m. from ∼175 cells analyzed from five independent experiments. **P≤0.01; ***P≤0.001 (unpaired two-tailed Student's t-test). (E) SHSY5Y cells were co-transfected with GFP-tagged MGRN1 or MGRN1ΔR and mitoRFP and imaged. Depletion of MGRN1 causes perinuclear clustering of mitochondria. Scale bars: 5 μm. (F) The mitochondrial distribution was calculated with ImageJ for cells imaged in E using their z-projections. Graph shows mean±s.e.m. from ∼120 cells analyzed from five independent experiments. **P≤0.01 (unpaired two-tailed Student's t-test). (G) Mitochondrial distribution in MEFs co-transfected with MGRN1 or MGRN1ΔR and mitoRFP. MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. The border of the transfected cell is marked by the dotted line. Scale bar: 5 μm.

Fig. 1.

Depletion of MGRN1 causes perinuclear clustering of mitochondria. (A) HeLa cells were treated with MGRN1 siRNA or mock siRNA or transfected with MGRN1 or MGRN1ΔR and imaged. Mitochondria were marked by mitoGFP. Note perinuclear clustering of mitochondria in cells upon the depletion of MGRN1. Images are 3D projections obtained from z-stacks using ImageJ; MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. Scale bars: 10 μm. The border of the transfected cell is marked by the dotted line. (B) The mitochondrial distribution was calculated with ImageJ for cells imaged in A using their z-projections. Graph shows mean±s.e.m. from ∼150 cells analyzed from five independent experiments. ***P≤0.001; ns, not significant (P=0.2) (unpaired two-tailed Student's t-test). (C) HeLa cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1 or MGRN1ΔR and mitoGFP after 48 h of siRNA treatment. z-stacks were taken 24 h later. Ectopic expression of MGRN1 but not MGRN1ΔR could rescue the mitochondrial clustering in MGRN1-depleted cells. MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. The border of the transfected cell is marked by the dotted line. Scale bars: 10 μm. (D) The mitochondrial distribution was calculated with ImageJ for cells imaged in C using their z-projections. Graph shows mean±s.e.m. from ∼175 cells analyzed from five independent experiments. **P≤0.01; ***P≤0.001 (unpaired two-tailed Student's t-test). (E) SHSY5Y cells were co-transfected with GFP-tagged MGRN1 or MGRN1ΔR and mitoRFP and imaged. Depletion of MGRN1 causes perinuclear clustering of mitochondria. Scale bars: 5 μm. (F) The mitochondrial distribution was calculated with ImageJ for cells imaged in E using their z-projections. Graph shows mean±s.e.m. from ∼120 cells analyzed from five independent experiments. **P≤0.01 (unpaired two-tailed Student's t-test). (G) Mitochondrial distribution in MEFs co-transfected with MGRN1 or MGRN1ΔR and mitoRFP. MGRN1 expression was verified by immunoblotting. The input levels of β-tubulin in the total lysates serve as loading controls. The border of the transfected cell is marked by the dotted line. Scale bar: 5 μm.

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To access the connectivity of the mictochondrial populations, HeLa cells were analyzed for fluorescence recovery after photobleaching (FRAP). The mitochondrial fluorescence recovered with a t1/2=∼30±3 s (mean±s.e.m., n=30) in control cells expressing MGRN1 (Fig. S2A,B). In contrast, little or no recovery was observed in MGRN1ΔR cells over a 5-min period, indicating that the mitochondrial network is compromised in the perinuclear clusters.

MGRN1 interacts with and ubiquitylates GP78, targeting it for proteasomal degradation

Given that overexpression of GP78 and depletion of MGRN1 similarly affected mitochondrial distribution, we checked whether MGRN1 could directly affect GP78 protein levels. HeLa cell lysates had significantly lower GP78 protein levels in the presence of functional MGRN1 than in cells expressing catalytically inactive RING mutants or vector controls. Corroborating this, higher levels of GP78 were present in MGRN1-depleted HeLa cells and melan md1-nc cells, compared to the corresponding controls (Fig. 2A,B). Ectopic overexpression of MGRN1 partially rescued the GP78 levels in cells treated with MGRN1 siRNAs (Fig. 2C). Because levels of GP78 decreased upon MGRN1 overexpression, we speculated that GP78 was itself an ubiquitylation substrate of MGRN1. MGRN1 co-immunoprecipitated with endogenous GP78 in mouse brain lysate (Fig. 2D,E) and with FLAG-tagged GP78 in HeLa cells (Fig. 2F,G). This interaction required the N-terminus of MGRN1 (Fig. 2H). Confocal imaging of cells expressing CyTERM–GFP (an ER marker; Costantini et al., 2012) and immunostained with anti-MGRN1 antibody showed that MGRN1, although chiefly cytosolic, did show some colocalization with the ER in HeLa cells (Fig. S2C). In addition, digitonin fractionation of cells showed that a minor fraction of MGRN1 was associated with membranes, although it was primarily cytosolic (Fig. S2D). This protein–protein interaction led to ubiquitylation of GP78, for which MGRN1 and ubiquitin were both required (Fig. 2I). Furthermore, in vivo ubiquitylation of FLAG–GP78 only occurred in the presence of MGRN1 and not MGRN1ΔR, demonstrating a requirement for functional MGRN1 (Fig. 2J). Depletion of MGRN1 reduced the polyubiquitylation smear (Fig. 2J). Among the various lysine mutants of ubiquitin, only transfection of K11 ubiquitin (a ubiquitin mutant with all lysine residues mutated to arginine except K11) was able to recapitulate a similar pattern of ubiquitylation to that seen with wild-type ubiquitin (Fig. 2J; Fig. S2E). Hence, MGRN1 caused K11-linked polyubiquitylation of GP78; GP78, however, did not affect the ubiquitylation of MGRN1 (Fig. 2K). Inhibiting the proteasome with MG132 restored the GP78 levels in the presence of MGRN1 to similar levels to those detected with catalytically inactive MGRN1 (MGRN1ΔR or C316D MGRN1) (Fig. 2L,M). To further verify this, GP78 protein levels in lysates from MGRN1- or MGRN1ΔR-expressing cells subjected to cycloheximide chase experiments were assayed. Drug treatment led to a decrease in GP78 levels over time in cells transfected with control vector or MGRN1, with faster kinetics observed in the presence of MGRN1 (Fig. 2N). Catalytic inactivation of MGRN1 substantially prolonged the half-life of GP78. Thus MGRN1-mediated ubiquitylation of GP78 regulates its steady-state levels.

Fig. 2.

MGRN1 interacts with and ubiquitylates GP78 for proteasomal degradation. (A) HeLa cells transfected with MGRN1 or the indicated RING mutants, or treated with mock or MGRN1 siRNAs were lysed and immunoblotted to check for the levels of GP78. Melanocytes, melan a-6 and melan md1-nc cell lysates were also analyzed for GP78. There are decreased GP78 protein levels in the presence of functional MGRN1. Control RFP vector (EmpVec) and MGRN1ΔR-transfected cells have comparable amounts of GP78. The input levels of β-tubulin and MGRN1 or RFP in the total lysates serve as loading controls. (B) Histogram plotting fold change in GP78 levels, analyzing data from A. Graph shows mean±s.e.m. from five independent experiments. **P≤0.01 (unpaired two-tailed Student's t-test). (C) Cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1, MGRN1ΔR or control vector (EmpVec) 48 h after siRNA treatment. Cells were lysed 24 h later and immunoblotted using anti-GP78 antibody. MGRN1 expression was verified by immunoblotting. (D) Mouse brain lysates were immunoprecipitated (IP) with anti-MGRN1 antibody. Western blot (IB) analysis of extract with anti-GP78 antibody shows co-immunoprecipitation of endogenous GP78 with MGRN1. Ab, antibody. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. (E) Reverse co-immunoprecipitation confirms the same result as in E. (F) HeLa cells co-transfected with FLAG-tagged GP78 and MGRN1–GFP were lysed, and immunoprecipitated with anti-MGRN1 antibody. Western blot analysis with anti-GP78 antibody shows co-immunoprecipitation of GP78 with MGRN1 when both proteins are overexpressed. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. (G) Reverse co-immunoprecipitation with HeLa cell lysates co-transfected with FLAG-tagged GP78 and MGRN1–GFP. (H) Line diagram of MGRN1 and its mutants used in this study. HeLa cells transiently co-transfected with FLAG–GP78 and the indicated GFP-tagged MGRN1 constructs were lysed and immunoprecipitated with anti-GFP antibody. Western blot analysis with anti-GP78 antibody shows co-immunoprecipitation of GP78 with MGRN1, MGRN1ΔR and MGRN1ΔC, but not with MGRN1ΔN. (I) HeLa cells transiently co-transfected with control RFP vector (EmpVec) or MGRN1–RFP and HA-tagged ubiquitin (Ub) constructs along with FLAG–GP78 were lysed and immunoprecipitated with anti-FLAG antibody (left panels). Cells transiently co-transfected with control HA vector (EmpVec) or HA-tagged Ub and MGRN1 constructs along with FLAG–GP78 were also similarly analyzed (right panels). In vivo ubiquitylation of GP78 was detected by immunoblotting for HA–Ub with anti-HA antibody. Polyubiquitylation is detected only when MGRN1 and Ub are both present. The input levels of β-tubulin, GP78 and MGRN1 or RFP in the total lysates serve as loading controls. (J) HeLa cells transiently co-transfected with HA–Ub, HA–K11Ub or HA–K11RUb constructs along with FLAG–GP78 and MGRN1–GFP or MGRN1ΔR–GFP were lysed and immunoprecipitated with anti-FLAG antibody. Cell treated with mock siRNA or MGRN1 siRNA were also similarly analyzed. In vivo ubiquitylation was detected by immunoblotting for HA–Ub with anti-HA antibody. Polyubiquitylation is detected in the presence of MGRN1 along with either Ub or K11Ub. The input levels of β-tubulin, MGRN1 and GP78 in the total lysates serve as loading controls. (K) HeLa cells transiently co-transfected with HA–Ub, MGRN1–GFP and FLAG-tagged GP78 or its RING domain mutant (RING MUT GP78) were lysed and immunoprecipitated with anti-GFP antibody. In vivo ubiquitylation of MGRN1–GFP was detected by immunoblotting for HA–Ub with anti-HA antibody. No significant difference in polyubiquitylation is detected between FLAG–GP78- and FLAG–GP78RINGmut-expressing cells. The input levels of MGRN1 and GP78 in the total lysates serve as loading controls. (L) Lysates from cells transiently transfected with MGRN1–GFP or indicated RING mutants were treated with proteasome inhibitor (20 µM MG132 for 4 h) or left untreated, followed by western blot analysis. Elevated levels of GP78 were detected upon MG132 treatment when MGRN1 is catalytically active. The input levels of β-tubulin and MGRN1 in the total lysates serve as a loading control. (M) Graph showing the mean±s.e.m. fold change in GP78 levels, analyzing data from L, from results of five independent experiments. *P≤0.05 (unpaired two-tailed Student's t-test). (N) Lysates from cells transiently transfected with control RFP vector (EmpVec), MGRN1–RFP or MGRN1ΔR–RFP were either left untreated or and treated with cycloheximide (Chx, 100 µg/ml) for the indicated periods of time. Western blot analyses show GP78 levels across samples. Note the decrease in protein levels over time in cells with EmpVec or MGRN1–RFP upon Chx treatment; the presence of MGRN1–RFP expedites the process. However, this rate is substantially slower in MGRN1ΔR–RFP-expressing cells.

Fig. 2.

MGRN1 interacts with and ubiquitylates GP78 for proteasomal degradation. (A) HeLa cells transfected with MGRN1 or the indicated RING mutants, or treated with mock or MGRN1 siRNAs were lysed and immunoblotted to check for the levels of GP78. Melanocytes, melan a-6 and melan md1-nc cell lysates were also analyzed for GP78. There are decreased GP78 protein levels in the presence of functional MGRN1. Control RFP vector (EmpVec) and MGRN1ΔR-transfected cells have comparable amounts of GP78. The input levels of β-tubulin and MGRN1 or RFP in the total lysates serve as loading controls. (B) Histogram plotting fold change in GP78 levels, analyzing data from A. Graph shows mean±s.e.m. from five independent experiments. **P≤0.01 (unpaired two-tailed Student's t-test). (C) Cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1, MGRN1ΔR or control vector (EmpVec) 48 h after siRNA treatment. Cells were lysed 24 h later and immunoblotted using anti-GP78 antibody. MGRN1 expression was verified by immunoblotting. (D) Mouse brain lysates were immunoprecipitated (IP) with anti-MGRN1 antibody. Western blot (IB) analysis of extract with anti-GP78 antibody shows co-immunoprecipitation of endogenous GP78 with MGRN1. Ab, antibody. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. (E) Reverse co-immunoprecipitation confirms the same result as in E. (F) HeLa cells co-transfected with FLAG-tagged GP78 and MGRN1–GFP were lysed, and immunoprecipitated with anti-MGRN1 antibody. Western blot analysis with anti-GP78 antibody shows co-immunoprecipitation of GP78 with MGRN1 when both proteins are overexpressed. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. (G) Reverse co-immunoprecipitation with HeLa cell lysates co-transfected with FLAG-tagged GP78 and MGRN1–GFP. (H) Line diagram of MGRN1 and its mutants used in this study. HeLa cells transiently co-transfected with FLAG–GP78 and the indicated GFP-tagged MGRN1 constructs were lysed and immunoprecipitated with anti-GFP antibody. Western blot analysis with anti-GP78 antibody shows co-immunoprecipitation of GP78 with MGRN1, MGRN1ΔR and MGRN1ΔC, but not with MGRN1ΔN. (I) HeLa cells transiently co-transfected with control RFP vector (EmpVec) or MGRN1–RFP and HA-tagged ubiquitin (Ub) constructs along with FLAG–GP78 were lysed and immunoprecipitated with anti-FLAG antibody (left panels). Cells transiently co-transfected with control HA vector (EmpVec) or HA-tagged Ub and MGRN1 constructs along with FLAG–GP78 were also similarly analyzed (right panels). In vivo ubiquitylation of GP78 was detected by immunoblotting for HA–Ub with anti-HA antibody. Polyubiquitylation is detected only when MGRN1 and Ub are both present. The input levels of β-tubulin, GP78 and MGRN1 or RFP in the total lysates serve as loading controls. (J) HeLa cells transiently co-transfected with HA–Ub, HA–K11Ub or HA–K11RUb constructs along with FLAG–GP78 and MGRN1–GFP or MGRN1ΔR–GFP were lysed and immunoprecipitated with anti-FLAG antibody. Cell treated with mock siRNA or MGRN1 siRNA were also similarly analyzed. In vivo ubiquitylation was detected by immunoblotting for HA–Ub with anti-HA antibody. Polyubiquitylation is detected in the presence of MGRN1 along with either Ub or K11Ub. The input levels of β-tubulin, MGRN1 and GP78 in the total lysates serve as loading controls. (K) HeLa cells transiently co-transfected with HA–Ub, MGRN1–GFP and FLAG-tagged GP78 or its RING domain mutant (RING MUT GP78) were lysed and immunoprecipitated with anti-GFP antibody. In vivo ubiquitylation of MGRN1–GFP was detected by immunoblotting for HA–Ub with anti-HA antibody. No significant difference in polyubiquitylation is detected between FLAG–GP78- and FLAG–GP78RINGmut-expressing cells. The input levels of MGRN1 and GP78 in the total lysates serve as loading controls. (L) Lysates from cells transiently transfected with MGRN1–GFP or indicated RING mutants were treated with proteasome inhibitor (20 µM MG132 for 4 h) or left untreated, followed by western blot analysis. Elevated levels of GP78 were detected upon MG132 treatment when MGRN1 is catalytically active. The input levels of β-tubulin and MGRN1 in the total lysates serve as a loading control. (M) Graph showing the mean±s.e.m. fold change in GP78 levels, analyzing data from L, from results of five independent experiments. *P≤0.05 (unpaired two-tailed Student's t-test). (N) Lysates from cells transiently transfected with control RFP vector (EmpVec), MGRN1–RFP or MGRN1ΔR–RFP were either left untreated or and treated with cycloheximide (Chx, 100 µg/ml) for the indicated periods of time. Western blot analyses show GP78 levels across samples. Note the decrease in protein levels over time in cells with EmpVec or MGRN1–RFP upon Chx treatment; the presence of MGRN1–RFP expedites the process. However, this rate is substantially slower in MGRN1ΔR–RFP-expressing cells.

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Depletion of MGRN1 alters mitofusin 1 protein levels but not mitochondrial mass

It has been reported that overexpression of functional GP78 leads to ubiquitylation of mitofusins, leading to their degradation (Fu et al., 2013). MGRN1-depleted cells phenocopied this result, and a decrease in mitofusin1 (Mfn1) levels was noted in cells expressing catalytically inactive MGRN1 (MGRN1ΔR or MGRN1C316D) and in cells treated with MGRN1 siRNA (Fig. 3A,B). Overexpression of MGRN1 did not alter the levels of Mfn1 or Mfn2 beyond that of the control cells. Levels of optic atrophy 1 (Opa1) decreased with functional depletion of MGRN1. The levels of other proteins regulating mitochondrial dynamics (Mfn2, Fis1 and Drp1) remained unchanged (Fig. 3C). The decrease in Mfn1 levels in MGRN1-knockdown cells could be rescued by expressing functional MGRN1 but not MGRN1ΔR (Fig. 3D). Mfn1 levels were less, but no detectable changes in Mfn2 levels could be seen, when MGRN1 was totally absent, as in melan md1-nc cells compared with the control melan a6 cells (Fig. 3E). GP78 has been reported to affect the protein levels of both mitofusins, but the effect of GP78 on Mfn1 protein levels is more prominent than that on Mfn2 (Fu et al., 2013), which might be the reason why indirect perturbation of GP78 by MGRN1 did not yield a detectable alteration in Mfn2 protein levels (Fig. S2F). Although perinuclear clustering of mitochondria and reduced levels of Mfn1 were observed with depletion of functional MGRN1, the overall mitochondrial mass remained unaffected. Equivalent levels of translocase of inner mitochondrial membrane 23 (Timm23) were detected across mitochondrial fractions isolated from MGRN1- and MGRN1ΔR-expressing cells (Fig. 3F). The mitochondrial DNA (mtDNA) levels with respect to the nuclear DNA (nDNA) were similar in both the samples (Fig. 3G). Therefore, as noted previously, GP78-mediated degradation of mitofusins occurs when GP78 levels are high, even in absence of CCCP, but this does not alter mitochondrial mass (Fu et al., 2013).

Fig. 3.

Depletion of MGRN1 causes decrease in Mfn1 levels but mitochondrial mass is unaltered. (A) Lysates from HeLa cells transiently transfected with the indicated constructs and immunoblotted show a decrease in Mfn1 protein levels upon MGRN1 functional depletion. Mfn2 levels remained unchanged. Levels of β-tubulin were used as the loading control, and the expression of MGRN1 and its mutants verified across different lysates. (B) Immunoblots from A were analyzed for the mean±s.e.m. fold change in Mfn1 and Mfn2 protein levels from five independent experiments. *P≤0.05; **P≤0.01; ns, not significant (P>0.1) (unpaired two-tailed Student's t-test). (C) Similar lysates generated as in A show that MGRN1 catalytic inactivation does not alter the protein levels of the fission mediators Fis1 and Drp1. Opa1 decreases with MGRN1 depletion. (D) HeLa cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1, MGRN1ΔR or control vector (EmpVec) 48 h after siRNA treatment. Cells were lysed 24 h later and immunoblotted using anti-Mfn1 antibody. Ectopic expression of MGRN1 rescues the decrease in Mfn1 level. The arrowhead indicates endogenous MGRN1. (E) Mfn2 protein remains unaltered in a-6 melan and md1-nc melan melanocyte cells lysates, whereas Mfn1 levels decrease in md1-nc melan melanocytes. Immunoblots from the top panel were analyzed for the mean±s.e.m. fold change in Mfn1 and Mfn2 protein levels from three independent experiments. **P≤0.01, ns, not significant (P=0.12) (unpaired two-tailed Student's t-test). (F) Mitochondria-enriched fractions from HeLa cells transfected with MGRN1 or MGRN1ΔR were immunoblotted using anti-Mfn1 and anti-Timm23 antibodies. Equal levels of Timm23 but lower levels of Mfn1 were detected in MGRN1ΔR-transfected cells. Expression of MGRN1 was verified in whole-cell lysates prior to fractionation. (G) Total DNA was isolated from MGRN1- or MGRN1ΔR-transfected cells. Quantitative RT-PCR was performed using Syber Green and primers against the mitochondrially encoded genes ATP synthase F0 and cytochrome c oxidase subunit II (COX-II) and the nuclear gene GAPDH. Samples were present in triplicates. ΔCt values for each mitochondrial DNA encoded gene was calculated as the Ct for the mitochondrial gene minus the Ct for GAPDH. ΔCt values do not differ significantly between MGRN1- and MGRN1ΔR-expressing cells. Error bars indicate s.d. ns, not significant (P=0.9).

Fig. 3.

Depletion of MGRN1 causes decrease in Mfn1 levels but mitochondrial mass is unaltered. (A) Lysates from HeLa cells transiently transfected with the indicated constructs and immunoblotted show a decrease in Mfn1 protein levels upon MGRN1 functional depletion. Mfn2 levels remained unchanged. Levels of β-tubulin were used as the loading control, and the expression of MGRN1 and its mutants verified across different lysates. (B) Immunoblots from A were analyzed for the mean±s.e.m. fold change in Mfn1 and Mfn2 protein levels from five independent experiments. *P≤0.05; **P≤0.01; ns, not significant (P>0.1) (unpaired two-tailed Student's t-test). (C) Similar lysates generated as in A show that MGRN1 catalytic inactivation does not alter the protein levels of the fission mediators Fis1 and Drp1. Opa1 decreases with MGRN1 depletion. (D) HeLa cells treated with MGRN1 siRNA or mock siRNA were transfected with MGRN1, MGRN1ΔR or control vector (EmpVec) 48 h after siRNA treatment. Cells were lysed 24 h later and immunoblotted using anti-Mfn1 antibody. Ectopic expression of MGRN1 rescues the decrease in Mfn1 level. The arrowhead indicates endogenous MGRN1. (E) Mfn2 protein remains unaltered in a-6 melan and md1-nc melan melanocyte cells lysates, whereas Mfn1 levels decrease in md1-nc melan melanocytes. Immunoblots from the top panel were analyzed for the mean±s.e.m. fold change in Mfn1 and Mfn2 protein levels from three independent experiments. **P≤0.01, ns, not significant (P=0.12) (unpaired two-tailed Student's t-test). (F) Mitochondria-enriched fractions from HeLa cells transfected with MGRN1 or MGRN1ΔR were immunoblotted using anti-Mfn1 and anti-Timm23 antibodies. Equal levels of Timm23 but lower levels of Mfn1 were detected in MGRN1ΔR-transfected cells. Expression of MGRN1 was verified in whole-cell lysates prior to fractionation. (G) Total DNA was isolated from MGRN1- or MGRN1ΔR-transfected cells. Quantitative RT-PCR was performed using Syber Green and primers against the mitochondrially encoded genes ATP synthase F0 and cytochrome c oxidase subunit II (COX-II) and the nuclear gene GAPDH. Samples were present in triplicates. ΔCt values for each mitochondrial DNA encoded gene was calculated as the Ct for the mitochondrial gene minus the Ct for GAPDH. ΔCt values do not differ significantly between MGRN1- and MGRN1ΔR-expressing cells. Error bars indicate s.d. ns, not significant (P=0.9).

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MGRN1-mediated ubiquitylation of GP78 is altered by mitochondrial stress

Given that high levels of GP78 regulate mitofusin protein level and affect mitochondrial mass in a CCCP-dependent manner (Fu et al., 2013), we hypothesized that the amount of GP78 protein in the cell might be regulated in a depolarization-dependent manner. We observed that the difference in GP78 levels between MGRN1- and MGRN1ΔR-transfected cells was reduced when cells were treated with CCCP (Fig. 4A). Next, we addressed whether the mitochondrial depolarization affected MGRN1-mediated regulation of GP78. MGRN1-mediated in vivo ubiquitylation of GP78 was severely compromised in the presence of mitochondrial stressors (like CCCP, antimycin A and oligomycin A). Vector controls lacked the CCCP sensitivity for the ubiquitylation (Fig. 4B). Furthermore, in cells expressing MGRN1 and GP78, the ubiquitylation signal intensity decreased with an increase in CCCP concentration (Fig. 4C). These results point to a mechanism where the interaction between MGRN1 and GP78 depends on mitochondrial health. As we showed above that the N-terminus of MGRN1 was essential for its interaction with GP78, we checked for the in vivo ubiquitylation of GP78 in the presence of MGRN1ΔN50 and MGRN1ΔN100 (N-terminal deletion mutants of MGRN1 lacking the first 50 or 100 amino acids, respectively) when cells were either treated with CCCP or left untreated. Ubiquitylation of GP78 in presence of MGRN1ΔN50 showed a mitochondrial stress dependence, like full-length MGRN1, but MGRN1ΔN100 did not (Fig. 4D). Ubiquitylation in presence of MGRN1ΔN100 was constitutive and did not change with CCCP treatment. This suggests that MGRN1ΔN100 does not interact with GP78. In this case, the ubiquitylation might be due to the effect of another E3 ligase that binds and post-translationally modifies GP78 when MGRN1 does not. Therefore amino acids 50–100 of MGRN1 interact with GP78 in a depolarization-dependent manner. It might be argued that, in the presence of MGRN1ΔR, association with GP78 would occur (through the first 50–100 amino acids of MGRN1) but that its ubiquitin-mediated degradation is compromised due to lack of the RING domain.

Fig. 4.

MGRN1-mediated ubiquitylation of GP78 is altered by mitochondrial stress. (A) HeLa cells transfected with MGRN1 or MGRN1ΔR were treated with CCCP (20 µM for 4 h) and immunoblotted with anti-GP78 antibody. Expression of MGRN1 was verified in cell lysates and β-tubulin was used as the loading control. (B) HeLa cells transiently co-transfected with HA-tagged ubiquitin (HA–Ub), FLAG–GP78 and MGRN1–GFP were either left untreated or treated with the indicated drugs. Lysates were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect HA–Ub-modified GP78. The blot shows a selective decrease in protein polyubiquitylation with drugs causing mitochondrial stress (left panels). Cells transfected with control vector were treated similarly (right panel). The input levels of MGRN1, β-tubulin and GP78 in the total lysates serve as loading controls. (C) HeLa cells co-transfected with MGRN1, FLAG–GP78 and HA–Ub were treated with the indicated concentrations of CCCP for 4 h. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody to detect GP78 ubiquitylated with HA–Ub. Ubiquitylation of GP78 decreases in a CCCP-concentration-dependent manner. The input levels of FLAG–GP78 and MGRN1 in the total lysates serve as loading control. (D) Line diagram of MGRN1 and its mutants. HeLa cells transiently expressing the indicated MGRN1 N-terminus deletion constructs along with HA–Ub and FLAG–GP78 were either treated with CCCP or left untreated; FLAG–GP78 was immunoprecipitated with anti-FLAG antibody. In vivo ubiquitylation of FLAG–GP78 was detected by immunoblotting with anti-HA antibody to detect HA–Ub-modified GP78. Note that CCCP partially abrogates GP78 polyubiquitylation in the presence of MGRN1ΔN50, but a similar effect is not detected with MGRN1ΔN100, which shows ubiquitylation irrespective of the presence of CCCP. (E) HeLa cells transiently expressing either MGRN1 or the indicated MGRN1 N-terminus deletion constructs along with HA–Ub, FLAG–GP78 were treated with CCCP (20 µM, 4 h) and BAPTA (75 µM, 4 h) in indicated combinations or left untreated; FLAG–GP78 was immunoprecipitated with anti-FLAG antibody. In vivo ubiquitylation was detected by immunoblotting with anti-HA antibody to detect HA–Ub. Note that BAPTA can partially rescue GP78 polyubiquitylation in CCCP-treated cells in the presence of MGRN1 and MGRN1ΔN50, but a similar effect is not detected with MGRN1ΔN100, which shows ubiquitylation irrespective of the presence of CCCP and BAPTA. (F) HeLa cells were treated with CCCP and BAPTA in the indicated combinations or left untreated (left graph). Fura-2AM was loaded and the cytosolic free Ca2+ concentration was measured from the ratio of fluorescence intensities obtained when samples were excited at 340 nm and 380 nm sequentially. Rmax and Rmin were calculated by digitonin permeabilization of Fura-2AM-loaded cells and by subsequent treatment with EGTA respectively. An apparent Kd for Fura-2-Ca was taken as 224 nM. An aliquot of cells transfected with the indicated MGRN1 constructs for the experiment in E (without drug treatment) were also similarly assayed for the free Ca2+ concentrations (right graph). Data represents mean±s.d. for three independent experiments with triplicates measured for each experiment. (G) HeLa cells co-transfected with FLAG–GP78 and MGRN1–RFP were lysed and immunoprecipitated in buffers containing either 5 mM CaCl2, no CaCl2 (control) or 5 mM MgCl2 together with 1 mM EGTA. MGRN1 was immunoprecipitated with anti-RFP antibody. Western blots analysis with anti-FLAG antibody shows co-immunoprecipitation of GP78 with MGRN1, in presence of low Ca2+ or no Ca2+, but the interaction is weaker in buffer supplemented with CaCl2. (H) HeLa cells co-transfected with MGRN1ΔN50–GFP or MGRN1ΔN100–GFP, and FLAG–GP78 were lysed and immunoprecipitated in buffers containing 5 mM CaCl2, no CaCl2 (control) or 5 mM MgCl2 together with 1 mM EGTA in a similar assay to that in G. MGRN1 is immunoprecipitated with anti-GFP antibody. Western blot analysis with anti-FLAG antibody shows that MGRN1ΔN50–GFP behaves in a similar manner to MGRN1, but the presence of MGRN1ΔN100 compromises the interaction between the two proteins. Note the lack of Ca2+-dependence in cells with MGRN1Δ100. (I) HeLa cells co-transfected with MGRN1 or MGRN1ΔR, RFP–LC3 and mitoGFP were treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h and imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). Arrowheads represent mitochondria in LC3-positive vesicles. The mean±s.e.m. number of LC3-positive mitochondria per cell is higher in MGRN1ΔR-expressing cells. Data represent five independent experiments with n=50 cells measured per experiment. ***P≤0.001 (unpaired two-tailed Student's t-test). (J) Cells expressing MGRN1 or MGRN1ΔR were treated with 20 µM CCCP for 4 h. Mitochondria-enriched fractions were immunoblotted using antibodies for LC3 and p62. Timm23 levels serve as loading control. (K) HeLa cells transfected with MGRN1 or MGRN1ΔR were loaded with TMRE and treated with CCCP. Time lapse images captured were analyzed for the mean±s.e.m. ratio of TMRE intensity at time ‘t’ (Ft) to the initial TMRE intensity (F0) and plotted against time. Data represent three independent experiments with n=20 cells measured per experiment. (L) HeLa cells transfected with different amounts of MGRN1 construct show a decrease in GP78 protein levels and an increase in MGRN1 protein expression. Mitochondria-enriched fractions from these cells show corresponding LC3 levels. Levels of β-tubulin and Timm23 serve as loading controls.

Fig. 4.

MGRN1-mediated ubiquitylation of GP78 is altered by mitochondrial stress. (A) HeLa cells transfected with MGRN1 or MGRN1ΔR were treated with CCCP (20 µM for 4 h) and immunoblotted with anti-GP78 antibody. Expression of MGRN1 was verified in cell lysates and β-tubulin was used as the loading control. (B) HeLa cells transiently co-transfected with HA-tagged ubiquitin (HA–Ub), FLAG–GP78 and MGRN1–GFP were either left untreated or treated with the indicated drugs. Lysates were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect HA–Ub-modified GP78. The blot shows a selective decrease in protein polyubiquitylation with drugs causing mitochondrial stress (left panels). Cells transfected with control vector were treated similarly (right panel). The input levels of MGRN1, β-tubulin and GP78 in the total lysates serve as loading controls. (C) HeLa cells co-transfected with MGRN1, FLAG–GP78 and HA–Ub were treated with the indicated concentrations of CCCP for 4 h. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody to detect GP78 ubiquitylated with HA–Ub. Ubiquitylation of GP78 decreases in a CCCP-concentration-dependent manner. The input levels of FLAG–GP78 and MGRN1 in the total lysates serve as loading control. (D) Line diagram of MGRN1 and its mutants. HeLa cells transiently expressing the indicated MGRN1 N-terminus deletion constructs along with HA–Ub and FLAG–GP78 were either treated with CCCP or left untreated; FLAG–GP78 was immunoprecipitated with anti-FLAG antibody. In vivo ubiquitylation of FLAG–GP78 was detected by immunoblotting with anti-HA antibody to detect HA–Ub-modified GP78. Note that CCCP partially abrogates GP78 polyubiquitylation in the presence of MGRN1ΔN50, but a similar effect is not detected with MGRN1ΔN100, which shows ubiquitylation irrespective of the presence of CCCP. (E) HeLa cells transiently expressing either MGRN1 or the indicated MGRN1 N-terminus deletion constructs along with HA–Ub, FLAG–GP78 were treated with CCCP (20 µM, 4 h) and BAPTA (75 µM, 4 h) in indicated combinations or left untreated; FLAG–GP78 was immunoprecipitated with anti-FLAG antibody. In vivo ubiquitylation was detected by immunoblotting with anti-HA antibody to detect HA–Ub. Note that BAPTA can partially rescue GP78 polyubiquitylation in CCCP-treated cells in the presence of MGRN1 and MGRN1ΔN50, but a similar effect is not detected with MGRN1ΔN100, which shows ubiquitylation irrespective of the presence of CCCP and BAPTA. (F) HeLa cells were treated with CCCP and BAPTA in the indicated combinations or left untreated (left graph). Fura-2AM was loaded and the cytosolic free Ca2+ concentration was measured from the ratio of fluorescence intensities obtained when samples were excited at 340 nm and 380 nm sequentially. Rmax and Rmin were calculated by digitonin permeabilization of Fura-2AM-loaded cells and by subsequent treatment with EGTA respectively. An apparent Kd for Fura-2-Ca was taken as 224 nM. An aliquot of cells transfected with the indicated MGRN1 constructs for the experiment in E (without drug treatment) were also similarly assayed for the free Ca2+ concentrations (right graph). Data represents mean±s.d. for three independent experiments with triplicates measured for each experiment. (G) HeLa cells co-transfected with FLAG–GP78 and MGRN1–RFP were lysed and immunoprecipitated in buffers containing either 5 mM CaCl2, no CaCl2 (control) or 5 mM MgCl2 together with 1 mM EGTA. MGRN1 was immunoprecipitated with anti-RFP antibody. Western blots analysis with anti-FLAG antibody shows co-immunoprecipitation of GP78 with MGRN1, in presence of low Ca2+ or no Ca2+, but the interaction is weaker in buffer supplemented with CaCl2. (H) HeLa cells co-transfected with MGRN1ΔN50–GFP or MGRN1ΔN100–GFP, and FLAG–GP78 were lysed and immunoprecipitated in buffers containing 5 mM CaCl2, no CaCl2 (control) or 5 mM MgCl2 together with 1 mM EGTA in a similar assay to that in G. MGRN1 is immunoprecipitated with anti-GFP antibody. Western blot analysis with anti-FLAG antibody shows that MGRN1ΔN50–GFP behaves in a similar manner to MGRN1, but the presence of MGRN1ΔN100 compromises the interaction between the two proteins. Note the lack of Ca2+-dependence in cells with MGRN1Δ100. (I) HeLa cells co-transfected with MGRN1 or MGRN1ΔR, RFP–LC3 and mitoGFP were treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h and imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). Arrowheads represent mitochondria in LC3-positive vesicles. The mean±s.e.m. number of LC3-positive mitochondria per cell is higher in MGRN1ΔR-expressing cells. Data represent five independent experiments with n=50 cells measured per experiment. ***P≤0.001 (unpaired two-tailed Student's t-test). (J) Cells expressing MGRN1 or MGRN1ΔR were treated with 20 µM CCCP for 4 h. Mitochondria-enriched fractions were immunoblotted using antibodies for LC3 and p62. Timm23 levels serve as loading control. (K) HeLa cells transfected with MGRN1 or MGRN1ΔR were loaded with TMRE and treated with CCCP. Time lapse images captured were analyzed for the mean±s.e.m. ratio of TMRE intensity at time ‘t’ (Ft) to the initial TMRE intensity (F0) and plotted against time. Data represent three independent experiments with n=20 cells measured per experiment. (L) HeLa cells transfected with different amounts of MGRN1 construct show a decrease in GP78 protein levels and an increase in MGRN1 protein expression. Mitochondria-enriched fractions from these cells show corresponding LC3 levels. Levels of β-tubulin and Timm23 serve as loading controls.

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Next, we addressed how this interaction between MGRN1 and GP78 could sense mitochondrial health. Given that treatment with CCCP, antimycin A or oligomycin A ultimately leads to an increased pool of cytosolic free Ca2+, it was logical to check whether this small molecule affected MGRN1-mediated GP78 ubiquitylation during mitochondrial stress. Moreover, Ca2+ is also an important molecule of crosstalk between the ER and mitochondria (de Brito and Scorrano, 2010). The in vivo ubiquitylation of GP78 was assayed in the presence of either CCCP alone or along with the Ca2+ chelator BAPTA. Presence of BAPTA could rescue the ubiquitylation of GP78 in CCCP-treated cells to give a similar level to that in untreated controls. Expression of MGRN1ΔN50 resulted in a similar phenotype to that seen upon expression of MGRN1; however, MGRN1ΔN100 showed constitutive ubiquitylation that did not change with either CCCP or BAPTA (Fig. 4E). Hence, it was prudent to hypothesize that the interaction between MGRN1 and GP78 could be dependent on cytosolic Ca2+. In that case MGRN1 and GP78 would interact when cytosolic Ca2+ was low, and high Ca2+ would disrupt such an interaction, eventually leading to reduced ubiquitylation and degradation of GP78. Free intracellular levels of Ca2+ levels were measured using Fura-2–acetoxymethyl-ester (FURA-2AM) in untreated control cells and in those treated with CCCP and/or BAPTA (Fig. 4F). Expression of MGRN1, MGRN1ΔN50 or MGRN1ΔN100 did not affect the levels of free intracellular Ca2+. Immunoprecipitation assays were performed to verify the interaction between GP78 and MGRN1 with different Ca2+ concentrations in a cell-free system. The association was strongest when EGTA was used to chelate Ca2+ and weakest when the buffer was supplemented with 5 mM CaCl2 (Fig. 4G). A similar Ca2+-dependent interaction was observed with MGRN1ΔN50 (Fig. 4H). Immunoprecipitation of GP78 by MGRN1 was compromised in the presence of MGRN1ΔN100 and was not affected by altering Ca2+ levels. These results suggest that MGRN1-mediated ubiquitylation modulates steady-state levels of GP78. This response does not occur upon an increase in cytosolic Ca2+ levels. Hence, MGRN1 indirectly participates in the mitochondrial quality control mechanism.

MGRN1-depleted cells show higher propensity for mitophagy

High levels of GP78 have been shown to increase mitophagy in a CCCP-dependent manner. Mitophagy events were quantified by analyzing the number of LC3-positive mitochondria from 3D projection images (Fig. S3A). No significant increase in sensitivity to CCCP was detected in control vector cells (Fig. S3B). When cells were co-transfected with MGRN1 or MGRN1ΔR, mitoGFP and RFP–LC3, and treated with low levels (1 µM) of CCCP and 100 nM bafilomycin A1, MGRN1ΔR-expressing cells revealed an increased number of LC3-positive mitochondria per cell compared to those expressing functional MGRN1 (Fig. 4I; see also Movies 3 and 4). Under similar drug treatments, MGRN1ΔR-expressing cells showed increased colocalisation of mitochondria with autophagic vesicles positive for p62 (also known as SQSTM1) than did the control MGRN1 cells (Fig. S3C,D). Even in the absence of either drug, cells with MGRN1ΔR had more LC3-positive mitochondria (Fig. S3B). Mitophagy was also monitored using a dual-tagged construct called mitoRosella (Rosado et al., 2008), which differentiates between neutral (white) healthy mitochondria and those in acidic (red) compartments (like amphisomes and autolysosomes). Similar to the results above, MGRN1ΔR-transfected cells had more mitochondria in acidic compartments per cell compared to MGRN1 control cells (Fig. S4A,B). Also, as reported previously (Fu et al., 2013), we found that GP78-mediated mitophagy was PARKIN independent; the cell lines in this study had a similar phenotypic distribution of mitochondria and levels of GP78 but drastically varied expression patterns of PARKIN (Fig. S4C). Hence, we propose that the mitophagy events observed upon functional depletion of MGRN1 are also PARKIN independent. The presence of MGRN1ΔR or non-functional MGRN1 increased the amount of LC3 and p62 in mitochondria-enriched fractions, even without CCCP treatment, suggesting that an increase in GP78 triggered a propensity for mitophagy (Fig. 4J; Fig. S3B). It was observed that catalytic inactivation of MGRN1 led to a higher propensity towards mitochondrial depolarization, as detected in cells loaded with the potentiometric dye tetramethylrhodamine ethyl ester (TMRE), followed by CCCP treatment (Fig. 4K). It was further verified that when cells were transfected with different amounts of the MGRN1 construct, GP78 levels decreased with the increase in MGRN1 protein levels. Mitochondria-enriched fractions from these cells showed a corresponding decrease in LC3 II (lipidated form of LC3 that is used as an autophagosomal marker) levels suggesting that an increase in MGRN1 leads to a decrease in GP78 that, in turn, decreases GP78-regulated mitophagy. Expression of MGRN1ΔR had the reverse effect on cellular GP78 and mitochondria-associated LC3 II levels (Fig. 4L). It has been previously reported that 10 µM CCCP treatment for 2 h is sufficient for recruitment of LC3 to the mitochondria-associated ER and detection of elevated levels of LC3 II in cells. However, for the evaluation of mitochondrial loss through mitophagy, a prolonged (24 h) treatment with CCCP is required (Fu et al., 2013). These results indirectly also suggest that high levels of GP78 prime mitochondria for mitophagy, but elevating its levels further by CCCP treatment or depolarization ultimately culminates in mitochondrial loss.

Collectively, our results point towards a mechanism in which MGRN1 keeps GP78 protein levels low in healthy cells but this regulation is withdrawn when the mitochondria is depolarized with CCCP. Mitochondrial stress, like that induced by CCCP, releases Ca2+ into the cytosol, and weakens the interaction between MGRN1 and GP78, causing an increase in GP78 levels that could then trigger mitophagy. Functional depletion of MGRN1 skews the balance towards depolarization and mitophagy. The increased propensity for mitophagy would also explain the decreased levels of the fusion protein Opa1 given that it has been previously reported that mitochondria destined for mitophagy are depolarized and lose Opa1 by degradation (Twig et al., 2008).

GP78 is downstream of MGRN1 during mitochondrial clustering and activation of mitophagy

Recent evidence shows that non-functional MGRN1 can block fusion between autophagosomes and lysosomes, but that the initial steps of autophagy are not affected by it. Maturation of late endosomes, generation of amphisomes and lysosomal proteolytic activity also does not get perturbed in these conditions (Majumder and Chakrabarti, 2015). Hence, we sought to confirm that the mitochondrial changes observed in MGRN1-depleted cells were due to GP78, and cells were co-transfected with different MGRN1 and GP78 constructs in the indicated combinations. Imaging studies showed that mitochondria clustered in the presence of functional GP78, irrespective of the MGRN1 status (Fig. 5A,B). Similarly Mfn1 levels were lower in cells where functional GP78 was overexpressed irrespective of the presence of MGRN1 (Fig. 5C). Also, with GP78 depletion, MGRN1ΔR overexpression no longer resulted in perinuclear clustering of mitochondria (Fig. 5D,E). To see whether the increase in mitophagy in MGRN1-depleted cells was mediated by GP78, we checked for mitophagy events in cells treated with GP78 siRNA and co-transfected with MGRN1 or MGRN1ΔR, mitoGFP and RFP–LC3. In cells treated with GP78 siRNA the difference between MGRN1- and MGRN1ΔR-expressing cells on the number of LC3-positive mitochondria was no longer significant (Fig. 5F). Thus, perinuclear clustering, and the decrease in Mfn1 levels and mitophagy might be attributable to GP78.

Fig. 5.

GP78 is downstream of MGRN1 during mitochondrial clustering and activation of mitophagy. (A) HeLa cells co-transfected with FLAG-tagged GP78 or its RING domain mutant (GP78 RINGmut), MGRN1 or MGRN1ΔR and mitoRFP were imaged under live-cell conditions. Altered mitochondrial distribution was detected upon overexpression of functional GP78, irrespective of the presence of MGRN1 or MGRN1ΔR. The transfected cell border is marked by the dotted line. Scale bars: 10 μm. (B) The histogram shows the mean±s.e.m. mitochondrial distribution for cells imaged in A. Results from five independent experiments. ns, not significant (P=0.4). (C) HeLa cells similarly transfected to those described in A were lysed and immunoblotted using anti-Mfn1 antibody. Overexpression of functional GP78 decreases Mfn1 levels irrespective of MGRN1. Levels of β-tubulin, GP78 and MGRN1–GFP serve as loading controls. (D) Cells treated with GP78 siRNA or mock siRNA, were co-transfected with MGRN1 or MGRN1ΔR, and mitoRFP were imaged for mitochondrial distribution. Note the well spread out mitochondria even in cells expressing MGRN1ΔR, when GP78 is depleted. The transfected cell border is marked by the dotted line. Scale bars: 10 μm. (E) Graph quantifying the mean±s.e.m. mitochondrial distribution for cells imaged in D. Results from five independent experiments. ***P≤0.001; ns, not significant (P=0.08) (unpaired two-tailed Student's t-test). (F) HeLa cells treated with GP78 siRNA or mock siRNA, co-transfected with MGRN1 and MGRN1ΔR, RFP–LC3 and mitoGFP and treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h, were imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). In GP78-knockdown cells the difference in mitophagy levels between MGRN1 and MGRN1ΔR becomes insignificant. Data represents the mean±s.e.m. for five independent experiments with n=50 cells measured per experiment. ***P≤0.001; ns, not significant (P=0.1) (unpaired two-tailed Student's t-test). Scale bars: 10 μm.

Fig. 5.

GP78 is downstream of MGRN1 during mitochondrial clustering and activation of mitophagy. (A) HeLa cells co-transfected with FLAG-tagged GP78 or its RING domain mutant (GP78 RINGmut), MGRN1 or MGRN1ΔR and mitoRFP were imaged under live-cell conditions. Altered mitochondrial distribution was detected upon overexpression of functional GP78, irrespective of the presence of MGRN1 or MGRN1ΔR. The transfected cell border is marked by the dotted line. Scale bars: 10 μm. (B) The histogram shows the mean±s.e.m. mitochondrial distribution for cells imaged in A. Results from five independent experiments. ns, not significant (P=0.4). (C) HeLa cells similarly transfected to those described in A were lysed and immunoblotted using anti-Mfn1 antibody. Overexpression of functional GP78 decreases Mfn1 levels irrespective of MGRN1. Levels of β-tubulin, GP78 and MGRN1–GFP serve as loading controls. (D) Cells treated with GP78 siRNA or mock siRNA, were co-transfected with MGRN1 or MGRN1ΔR, and mitoRFP were imaged for mitochondrial distribution. Note the well spread out mitochondria even in cells expressing MGRN1ΔR, when GP78 is depleted. The transfected cell border is marked by the dotted line. Scale bars: 10 μm. (E) Graph quantifying the mean±s.e.m. mitochondrial distribution for cells imaged in D. Results from five independent experiments. ***P≤0.001; ns, not significant (P=0.08) (unpaired two-tailed Student's t-test). (F) HeLa cells treated with GP78 siRNA or mock siRNA, co-transfected with MGRN1 and MGRN1ΔR, RFP–LC3 and mitoGFP and treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h, were imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). In GP78-knockdown cells the difference in mitophagy levels between MGRN1 and MGRN1ΔR becomes insignificant. Data represents the mean±s.e.m. for five independent experiments with n=50 cells measured per experiment. ***P≤0.001; ns, not significant (P=0.1) (unpaired two-tailed Student's t-test). Scale bars: 10 μm.

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Functional depletion of MGRN1 by disease-causing PrP mutants affects ubiquitylation of GP78

It has been suggested that MGRN1 interacts with an aberrant metabolic isoform of the ubiquitously expressed cell surface glycoprotein, mammalian PrP, referred to as CtmPrP. Studies have shown that increased generation of CtmPrP {either by expression of artificial constructs, like PrP(AV3) or PrP(KH-II), or through naturally occurring human disease mutations [PrP(A117V)]} leads to spongiform neurodegeneration in animal models (Hegde et al., 1998; Rane et al., 2010) and also affects the activity of MGRN1 in cell culture systems (Chakrabarti and Hegde, 2009). Brain lysates from transgenic mice expressing CtmPrP [PrP(A117V)] showed a decrease in Mfn1 and increase in GP78 levels, whereas Mfn2 protein levels remained unaltered, when compared with the non-transgenic control (Fig. 6A). In HeLa cells, expression of the indicated CtmPrP-generating constructs [PrP(AV3), PrP(KHII) or PrP(A117V)] also resulted in similar changes in the protein levels of Mfn1 and GP78 (Fig. 6B). Wild-type PrP-expressing cells had comparable levels of Mfn1 protein to those of cells expressing the empty vector control (data not shown). In transiently transfected cultured cells expressing wild-type PrP, <1–2% of the protein is present as CtmPrP, this percentage, however, increases in the presence of the above mutants (Chakrabarti and Hegde, 2009). Polyubiquitylation of GP78 was severely compromised in the presence of CtmPrP, indicating that even indirect perturbation of MGRN1 activity is sufficient to elicit an effect similar to its depletion (Fig. 6C). Simultaneously, increased levels of GP78 (due to lack of its polyubiquitylation and clearance) led to perinuclear mitochondrial clusters in CtmPrP-expressing cells (Fig. 6D). The CCCP dependence of GP78 polyubiquitylation was evident in the presence of PrP (given that this does not functionally perturb MGRN1), but not with PrP(A117V) (Fig. 6E). The presence of CtmPrP led to an increase in LC3 II and p62 at the mitochondria similar to with MGRN1ΔR (Fig. 6F). A higher number of LC3-positive mitochondria were detected in PrP(KHII)-expressing cells, where there is less functional MGRN1 than in those expressing PrP (Fig. 6G). Taking all these results into consideration, it can be hypothesized that MGRN1 plays a role in regulating mitochondrial turnover by regulating GP78 in a subset of familial prion diseases, and that it might contribute to CtmPrP-mediated neurodegeneration.

Fig. 6.

Functional depletion of MGRN1 by ectopic expression of PrP mutants perturbs ubiquitylation of GP78 and mitophagy. (A) Whole-brain lysates from transgenic mice were immunoblotted and analyzed for the indicated proteins. The levels of β-tubulin serve as loading controls. Note similar levels of expression of PrP and MGRN1 across samples. (B) Samples prepared from HeLa cells transiently transfected with wild-type PrP and CtmPrP-generating mutants were lysed and immunoblotted to check for the levels of Mfn1, Mfn2 and GP78. The levels of β-tubulin served as a loading control. (C) Lysates of cells expressing with HA-tagged ubiquitin (Ub) and FLAG–GP78 along with PrP constructs were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect GP78 ubiquitylated with HA–Ub. The blot shows a selective decrease in protein polyubiquitylation in the presence of CtmPrP-generating mutants. The input levels of PrP, β-tubulin and GP78 in the total lysates serve as a loading control. (D) Cells co-transfected with wild-type PrP or the indicated PrP mutants and mitoRFP were imaged under live-cell conditions. Perinuclear mitochondrial clustering detected upon expression of the CtmPrP-generating mutants. Scale bar: 10 μm. (E) HeLa cells transiently co-transfected with HA–Ub, FLAG–GP78 and PrP(A117V) or PrP were either left untreated or treated with CCCP. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody to detect HA–Ub in vivo ubiquitylation. The blot shows a selective decrease in protein polyubiquitylation with CCCP when PrP is present. The input levels of PrP, β-tubulin and GP78 in the total lysates serve as loading controls. (F) Cells transfected with the indicated PrP constructs were treated with 100 nM Bafilomycin A1 for 16 h. Mitochondria-enriched fractions were immunoblotted using antibodies for LC3 and p62. Timm23 levels serve as loading control. (G) HeLa cells co-transfected with wild-type PrP or PrP(KHII), RFP–LC3 and mitoGFP were treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h and imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). Arrowheads represent mitochondria in LC3-positive vesicles. The number of LC3-positive mitochondria per cell is higher in PrP(KHII)-expressing cells. Data represents the mean±s.e.m. for five independent experiments with n=50 cells measured per experiment. ***P≤0.001 (unpaired two-tailed Student's t-test). Scale bars: 10 μm.

Fig. 6.

Functional depletion of MGRN1 by ectopic expression of PrP mutants perturbs ubiquitylation of GP78 and mitophagy. (A) Whole-brain lysates from transgenic mice were immunoblotted and analyzed for the indicated proteins. The levels of β-tubulin serve as loading controls. Note similar levels of expression of PrP and MGRN1 across samples. (B) Samples prepared from HeLa cells transiently transfected with wild-type PrP and CtmPrP-generating mutants were lysed and immunoblotted to check for the levels of Mfn1, Mfn2 and GP78. The levels of β-tubulin served as a loading control. (C) Lysates of cells expressing with HA-tagged ubiquitin (Ub) and FLAG–GP78 along with PrP constructs were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect GP78 ubiquitylated with HA–Ub. The blot shows a selective decrease in protein polyubiquitylation in the presence of CtmPrP-generating mutants. The input levels of PrP, β-tubulin and GP78 in the total lysates serve as a loading control. (D) Cells co-transfected with wild-type PrP or the indicated PrP mutants and mitoRFP were imaged under live-cell conditions. Perinuclear mitochondrial clustering detected upon expression of the CtmPrP-generating mutants. Scale bar: 10 μm. (E) HeLa cells transiently co-transfected with HA–Ub, FLAG–GP78 and PrP(A117V) or PrP were either left untreated or treated with CCCP. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody to detect HA–Ub in vivo ubiquitylation. The blot shows a selective decrease in protein polyubiquitylation with CCCP when PrP is present. The input levels of PrP, β-tubulin and GP78 in the total lysates serve as loading controls. (F) Cells transfected with the indicated PrP constructs were treated with 100 nM Bafilomycin A1 for 16 h. Mitochondria-enriched fractions were immunoblotted using antibodies for LC3 and p62. Timm23 levels serve as loading control. (G) HeLa cells co-transfected with wild-type PrP or PrP(KHII), RFP–LC3 and mitoGFP were treated with 1 μM CCCP and 100 nM Bafilomycin A1 for 16 h and imaged to observe mitophagy events. Enlarged views of the areas within the white boxes are also shown (insets). Arrowheads represent mitochondria in LC3-positive vesicles. The number of LC3-positive mitochondria per cell is higher in PrP(KHII)-expressing cells. Data represents the mean±s.e.m. for five independent experiments with n=50 cells measured per experiment. ***P≤0.001 (unpaired two-tailed Student's t-test). Scale bars: 10 μm.

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This study elucidates a new interaction of the cytosolic E3 ligase MGRN1 with the ER ubiquitin E3 ligase GP78. This results in trans ubiquitylation through non-canonical K11 linkages and degradation of GP78. The reverse is not true, meaning GP78 is not responsible for the regulation of MGRN1. Functional MGRN1 constitutively maintains low levels of GP78; this subjugates basal mitophagy in healthy cells. When mitochondria are stressed and cytosolic Ca2+ increases, the ubiquitylation of GP78 mediated by MGRN1 gets compromised. Reduced ubiquitylation is due to a diminished interaction between MGRN1 and GP78 in the presence of high levels of cytosolic Ca2+. GP78 interacts with the N-terminus of MGRN1 (more precisely amino acids 50–100) and this results in Ca2+-dependent ubiquitylation. Chelating away Ca2+ restores the association of GP78 with MGRN1 in a cell-free system; likewise in trans in vivo ubiquitylation of GP78 also occurs in the presence of BAPTA. Lack of interaction between GP78 and MGRN1 hence overrides the Ca2+ dependence of this trans ubiquitylation. Phenotypically the decreased MGRN1-mediated ubiquitylation and degradation of GP78 in the presence of mitochondrial stress is reflected as an increase in propensity for mitophagy (Fig. 7). Elevated levels of GP78 and the consequential increase in the initiation of mitophagy is further detected when MGRN1 is catalytically inactive. This study becomes more important because perturbation of MGRN1 function in the presence of disease-causing CtmPrP mutants compromises trans polyubiquitylation of GP78. Here again, CCCP-dependent regulation of GP78 levels is observed only in the presence of PrP and not CtmPrP. Increased mitophagy is detected with overexpression of CtmPrP in cells. This study, hence, highlights how MGRN1 detects mitochondrial stress and participates in mitochondrial biogenesis. It is perfectly plausible to extrapolate that non-functional MGRN1 could contribute to neurodegeneration in at least a subset of CtmPrP-mediated prion diseases.

Fig. 7.

Schematic diagram summarizing the results. MGRN1-mediated polyubiquitylation of GP78 occurs in healthy cells where cytosolic Ca2+ levels are low. This regulates GP78 protein levels. When cytosolic Ca2+ rises (or when MGRN1 is nonfunctional) the interaction between MGRN1 and GP78 weakens, leading to elevated GP78 levels and an increase in GP78-mediated mitophagy.

Fig. 7.

Schematic diagram summarizing the results. MGRN1-mediated polyubiquitylation of GP78 occurs in healthy cells where cytosolic Ca2+ levels are low. This regulates GP78 protein levels. When cytosolic Ca2+ rises (or when MGRN1 is nonfunctional) the interaction between MGRN1 and GP78 weakens, leading to elevated GP78 levels and an increase in GP78-mediated mitophagy.

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Cells have evolved rigorous surveillance systems to efficiently detect and eliminate damaged and dysfunctional mitochondria. An interdependent but hierarchical cellular quality control mechanism exists to maintain normal mitochondrial biogenesis and to ensure cell survival (Rugarli and Langer, 2012). Chaperones and proteases inside the mitochondria are responsible for proper folding of pre-proteins imported into the organelle and degradation of irreversibly damaged polypeptides – these constitute part of the intraorganellar quality control system (Tatsuta and Langer, 2008). The mitochondrial unfolded protein response (mtUPR), part of this quality control mechanism, is closely associated with the nuclear transcriptional program that induces the expression of mitochondrial chaperones and proteases under conditions of mitochondrial stress (Zhao et al., 2002; Benedetti et al., 2006). This first line of defense is closely followed by the organellar quality control system, which alters the balance between fission and fusion during stress. For example, nutrient-deprivation-induced stress or stalling cytosolic protein synthesis might lead to impaired fission and unhindered mitochondrial fusion (Tondera et al., 2009; Gomes et al., 2011; Rambold et al., 2011; Youle and van der Bliek, 2012). By contrast, this balance is pushed more towards fission when mitochondria are depolarized or there is excessive reactive oxygen species (ROS) generation in these organelles. This ultimately culminates in elimination of dysfunctional mitochondria through mitophagy (Twig et al., 2008; Narendra et al., 2008; Wang et al., 2012; Frank et al., 2012).

It is well established that the cytosolic ubiquitin E3 ligase PARKIN is recruited to damaged mitochondria by PTEN-induced putative kinase 1 (PINK1) and targets them for mitophagy. This happens when these organelles are depolarized and there is increased uncoupling of mitochondria (Matsuda et al., 2010; Gegg et al., 2010; Narendra et al., 2010; Youle and Narendra, 2011; Ziviani et al., 2010). Once localized to the mitochondria, PARKIN ubiquitylates VDAC1 and recruits the autophagic adapter p62 to facilitate mitophagic clearance of these dysfunctional organelles (Geisler et al., 2010). Upon depolarization, PARKIN mediates mitochondrial localization of p97 (also known as VCP), an AAA-ATPase involved in the retrotranslocation of ER-membrane-spanning proteins after ubiquitylation en route to proteasome (Ye et al., 2001; Rabinovich et al., 2002; Tanaka et al., 2010). Furthermore, PINK-phosphorylated Mfn2 might also act as a PARKIN receptor on depolarized mitochondria (Chen and Dorn, 2013). The altered localization of PARKIN and p97 during mitochondrial stress results in ubiquitylation and proteasomal degradation of Mfn1 and Mfn2, which in turn triggers mitophagy (Tanaka et al., 2010). These findings illustrate that PARKIN primarily facilitates mitochondrial quality control at the organellar level. By contrast, the mitochondrial E3 ubiquitin ligase MUL1 is transcriptionally upregulated under mitochondrial stress through a mechanism involving FoxO1 and FoxO3 transcription factors (Lokireddy et al., 2012). Elevated levels of MUL1 lead to the ubiquitylation and proteasomal degradation of Mfn2, finally culminating in mitophagy (Lokireddy et al., 2012; Yun et al., 2014). Either relocation of a cytosolic ligase to the mitochondria or transcriptional upregulation of a mitochondrial ligase under stress helps recruit the molecular components required for mitophagy. These findings strengthen the argument that mechanisms must inherently exist in cells to distinguish normal and stress conditions and facilitate efficient differential activity of these enzymes.

The ER-associated E3 ubiquitin ligase GP78 also extends support to the mitochondrial homoeostasis through organellar quality control. This ligase is an integral component of the ERAD machinery, whereby misfolded proteins inside the ER are ubiquitylated and retro-translocated to the cytosol for proteasomal degradation (Fang et al., 2001; Fairbank et al., 2009). Although high levels of GP78 constitutively interacts with, ubiquitylates and degrades Mfn1 (primarily) and Mfn2 (a more modest effect), it regulates mitophagy only upon mitochondrial depolarization (Fu et al., 2013). It is plausible to hypothesize that GP78 levels should be maintained at low levels in cells under normal circumstances to prevent unregulated degradation of mitofusins. Simultaneously, these results also emphasize the need for a molecular sensor to detect depolarization of mitochondria, during which GP78-mediated degradation of mitofusins and the onset of mitophagy would be physiologically beneficial. Our present study, identifying polyubiquitylation and degradation of GP78 by MGRN1 preferentially under normal circumstances, thus provides a mechanism to prevent this ERAD-associated protein from indiscriminately removing mitofusins from cells. This might serve a dual purpose of also keeping a check on ERAD.

It is well established that many protein–protein interactions are affected by cytosolic Ca2+ levels (Vito et al., 1996; Missotten et al., 1999; Park et al., 2013). Our results indicate that under conditions of high cytosolic Ca2+, the interaction between MGRN1 and GP78 is disrupted and this prevents ubiquitylation and degradation of GP78. Given that the physical association between MGRN1 and GP78 is key for this post-translational modification and eventual deregulation, functional sequestration of MGRN1 by disease-causing CtmPrP mutants elicits similar results to the presence of high cytosolic Ca2+ – thus, mitochondrial deregulation affected by the ubiquitously expressed MGRN1 could be one of the factors governing neurodegeneration in a subset of prion diseases.

The effect of Ca2+ might be direct, where MGRN1 can detect fluctuating Ca2+ levels, or indirect, where MGRN1 competes with another ligase for binding and ubiquitylating GP78. The association of the unknown ligase is stronger in the presence of Ca2+ and hence this competes out MGRN1. Further insight into the mechanism might come from a better understanding of the regulation of GP78 under physiological conditions.

Furthermore, an involvement of Ca2+-dependent regulation in the regulation of ER–mitochondrial contacts by GP78 has been previous reported (Wang et al., 2000,, 2015; Goetz et al., 2007; Li et al., 2015). Presence of autocrine motility factor (AMF, also known as GPI) or the RING mutant GP78 has been shown to decrease ER–mitochondria contacts and increase ER–mitochondria Ca2+ coupling times upon ATP stimulation (Wang et al., 2015). Depletion of mitofusins leads to enhanced ER–mitochondria Ca2+ crosstalk, and increased sensitivity to mitochondrial-Ca2+-mediated cell death (Filadi et al., 2015; Wang et al., 2015). Also, post-translational modification of GP78 (phosphorylation) has been shown to affect GP78-dependent mitofusin degradation (Li et al., 2015).

It is possible to extrapolate that MGRN1-mediated ubiquitylation of GP78 might be another layer of regulation to this complex system that phenocopies the effect of AMF or RING mutant GP78 on ER–mitochondria contacts and Ca2+ coupling. When cytosolic Ca2+ levels are high, MGRN1–GP78 interactions weaken, which might cause increase in ER–mitochondria contacts, favoring GP78-dependent degradation of mitofusins and mitophagy. Decreased levels of mitofusins in high cytosolic Ca2+ conditions might also predispose cells to other cell death pathways, like apoptosis. These hypotheses, however, need to be experimentally substantiated.

Excess cytosolic Ca2+ might in turn also lead to ER stress. Under such circumstances it would be logical to have higher GP78 levels for an efficient ERAD system. We suggest that MGRN1 provides another layer of complexity to the already existing multi-tiered quality control mechanism by repressing GP78 in healthy cells.

Constructs, antibodies and reagents

MGRN1, MGRN1ΔR, MGRN1ΔN, MGRN1ΔC, C316DMGRN1, PrP, PrP(A117V), PrP(KHII), PrP(AV3) are as described previously (Chakrabarti and Hegde, 2009; Srivastava and Chakrabarti, 2014). HA-tagged wild-type ubiquitin was a gift from Rafael Mattera (Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD); K0, K48 and K63 ubiquitin mutants were gifts of Kah-Leong Lim (Neurodegeneration Research Laboratory, National Neuroscience Institute, Singapore); HA-tagged K6, K11, K29 and K11R ubiquitin mutants were gifts of Tomohiko Ohta (Department of Translational Oncology, St. Marianna University, Kanagawa, Japan). K6, K11, K29, K48 and K63 ubiquitin mutants are lysine mutants of ubiquitin where all other lysines are mutated to arginine except for the residue in the name. K0 ubiquitin mutant is a lysine-less ubiquitin, only capable of mediating monoubiquitylation. The K11R ubiquitin mutant has only lysine 11 mutated to arginine. FLAG-tagged GP78, FLAG-tagged RING mutant GP78, GP78–GFP and GP78-RING-mutant–GFP were gifts of Ivan Nabi (Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada). mito-Rosella was a gift from Rodney Devenish (Department of Biochemistry and Molecular Biology, Monash University, Australia), CyTERM–GFP was a gift from Erik Snapp (Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, NY, USA), GFP–LC3 was a gift from Debashis Mukhopadhyay (Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India), pAcGFP1-Mito (mitoGFP) was a gift from Subrata Banerjee (Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India), RFP–LC3, mitoRFP, MGRN1ΔN50 and MGRN1ΔN100 were generated using standard cloning techniques.

Antibodies were against the following proteins: GP78 (#sc-166358, Santa Cruz Biotechnology, 1:250), Mfn1 (#ab57602, Abcam, Cambridge, UK; 1:1000), Mfn2 (#ab124773, Abcam, Cambridge, UK; 1:1000), Opa1 (#MA5-16149, Thermo Scientific, Rockford, IL; 1:1000), Fis1 (#PA22142, Thermo Scientific, Rockford, IL, USA; 1:1000), Drp1 (#ab56788, Abcam, Cambridge, UK; 1:1000) COX 4 (#4844S, Cell Signaling Technology; 1:100), ATP synthase complex V (#459240, Invitrogen, CA; 1:1000), β-tubulin (#ab7792, Abcam, Cambridge, UK; 1:2000), Timm 23 (#ab116329, Abcam, Cambridge, UK; 1:2000) p62/SQSTM1 (#PA20839, Thermo Scientific, Rockford, IL; 1:2000), LC3 (#NB100-2220, Novus Biologicals; 1:2000) and ubiquitin (#U5379, Sigma-Aldrich; 1:1000). The anti-MGRN1, -GFP, -RFP, -TRAPα, -Myc, -FLAG and -HA antibodies were gifts of Ramanujan S. Hegde (MRC Laboratory of Molecular Biology, Cambridge, UK) and were used at 1:3000.

MG132, CCCP, oligomycin A, antimycin A, FURA-2AM and BAPTA-AM were from Sigma-Aldrich; proteinase K was from GIBCO-BRL; Mitotracker Deep Red FM was from Molecular Probes, Invitrogen; and Universal FastStart Syber Green Master (Rox) was from Roche. Drug treatments used in the study were as follows: MG132 (20 µM, 4 h), cycloheximide (100 µg/ml), CCCP (20 µM, 4 h), oligomycin A (10 µM, 2 h), antimycin A (10 µM, 6 h), BAPTA-AM (75 µM, 4 h) and Bafilomycin A1 (100 nM, 16 h).

Cell culture, transfection and siRNA-mediated knockdown

Cell lines used for the experiments were HeLa cells (human cervical cancer cell line), SH-SY5Y cells (human neuroblastoma cell line), MEF cells (mouse embryonic fibroblast cells), immortal melanocytes (control melan-a6 melanocytes or the Mgrn1-null melan md1-nc melanocytes). Maintenance of HeLa cells in culture was as described previously (Srivastava and Chakrabarti, 2014). Briefly, cells were grown in 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) in Dulbecco's modified Eagle's medium (DMEM; Himedia, Mumbai, India) at 37°C and 5% CO2. Immortal melanocytes were grown in culture as described previously (Srivastava and Chakrabarti, 2014; Hida et al., 2009). SH-SY5Y cells (a gift from Debashis Mukhopadhyay) and MEF cells (CF-1 strain; a gift from Mitradas M. Panicker, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India) were also grown under standard cell culture conditions as described previously (Srivastava and Chakrabarti, 2014; Roy et al., 2013). Immortal melanocytes (a gift from Ramanujan S. Hegde) were obtained from the Wellcome Trust Functional Genomics Cell Bank.

For transfections of cells, Lipofectamine 2000 was used (Invitrogen) as per the manufacturer's instructions. At 24 h after transfection, cells were lysed using suitable lysis buffer. For all siRNA-mediated knockdowns, pooled siRNAs (Dharmacon ON-TARGETplus SMARTpool) were used, consisting of a mixture of four individual siRNAs. siRNA pools used in the study are as follows: MGRN1 (L-022620-00-0020), GP78 (L-006522-00-0010), and non-targeting siRNA (D-001810-01-20).

All tissue culture plastic-ware and Lab-Tek 8-well chambered slides used for microscopy were from Nunc, confocal 35-mm clear-coverglass-bottom dishes used for microscopy were from SPL Lifesciences.

Mitotracker Deep Red staining and immunocytochemistry

Mitotracker Deep Red FM was loaded in HeLa cells by incubating live cells with 100 nM dye for 20 min followed by fixation with 4% formaldehyde and immunostaining. For immunocytochemistry, cells were fixed with either 4% formaldehyde or methanol as per the requirement of the antibody, as described previously (Chakrabarti and Hegde, 2009; Srivastava and Chakrabarti, 2014). Cells were permeabilized using 10% FBS in PBS with 0.1% saponin (Sigma-Aldrich) for 60 min, followed by overnight staining in primary antibody at 4°C and a 60-min incubation in secondary antibody at room temperature. The samples were then imaged with a confocal microscope.

Western blotting and immunoprecipitation

The protocol for western blotting was as described previously (Srivastava and Chakrabarti, 2014). 10% Tris-tricine gels or 7.5% Tris glycine gels were used for SDS-PAGE followed by western blotting. Quantification of western blots was performed with Quantity One software (Bio-Rad). At least three independent experiments were performed and band intensities were normalized to that of the loading control. P-values were determined using Student's t-test. For immunoprecipitation, cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1% IGEPAL, 1 mM PMSF and protease inhibitor cocktail (Sigma-Aldrich)], and immunoprecipitation was performed under denaturing conditions as described previously (Srivastava and Chakrabarti, 2014).

Total DNA isolation and quantitative RT-PCR

Total DNA was isolated by lysing HeLa cells from a 90-mm dish in 0.5 ml lysis buffer (10 mM Tris-HCl pH 8.0, 0.1 M EDTA and 0.1% SDS). 10 μl proteinase K (200 mg/ml) was added and incubated at 55°C for 3 h. The tubes were then centrifuged at 8000 g for 15 min. To the supernatant, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, followed by centrifugation at 8000 g for 15 min. To the aqueous phase, an equal volume of chloroform was added, followed by centrifugation at 8000 g for 15 min. The DNA was precipitated from the aqueous phase by adding one volume of isopropanol and a one-tenth volume of 3 M sodium acetate, followed by centrifugation at 8000 g for 15 min. The pellet was washed in 70% ethanol and resuspended in 0.4 ml Tris EDTA buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). 100 ng of this DNA was used as a template in RT-PCR using Syber Green and primers against the mitochondrially encoded genes ATPsynthase F0 and cytochrome c oxidase subunit II (COXII) and the nuclear encoded gene GAPDH. Primer sequences are as follows: cytochrome oxidase subunit II, COX II Fwd, 5′-ATCAAATCAATTGGCCACCAATGGTA-3′ and COXIIRev, 5′-TTGACCGTAGTATACCCCCGGTC-3′; ATPsynthase F0, ATPS Fwd, 5′-TTTCCCGCTCTATTGATCCC-3′ and ATPS Rev, 5′-GATGGCCATGGCTAGGTTTA-3′; and GAPDH, GAPDH Fwd, 5′-AGAAGGCTGGGGCTCATTTG-3′ and GAPDH Rev, 5′-AGGGGCCATCCACAGTCTTC-3′.

In vivo ubiquitylation assay

The in vivo ubiquitylation assay was performed as previously described (Srivastava and Chakrabarti, 2014). Briefly, cells co-transfected with HA-tagged wild-type ubiquitin (or ubiquitin mutants), MGRN1 and FLAG- or GFP-tagged GP78 constructs were lysed in immunoprecipitation buffer and immunoprecipitated under denaturing condition with anti-FLAG or anti-GFP antibody. Ubiquitylated GP78 or MGRN1 was detected by immunoblotting with anti-HA antibody. Ubiquitin constructs used are as previously described (Mattera et al., 2004; Nishikawa et al., 2004; Tan et al., 2008).

Digitonin fractionation

HeLa cells were lysed in KHM buffer with digitonin (20 mM Hepes pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate and 100 µg/ml digitonin) followed by centrifugation at 2000 g to separate membrane and cytosolic fractions. The membrane fraction was then washed with KHM buffer and resuspended in KHM buffer with NP-40 (20 mM Hepes pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate and 1% NP-40). SDS-PAGE was performed followed by immunoblotting with antibodies against MGRN1, GP78, GAPDH and TRAPα.

Subcellular fractionation to obtain mitochondria-enriched fractions

HeLa cells were lysed in isolation buffer (20 mM Hepes pH 7.4, 10 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF and 0.25 M sucrose) by passing through a 25 gauge needle attached on a 1-ml syringe ten times. This was centrifuged at 600 g to pellet unlysed cell debris and nuclear fractions. The supernatant was then centrifuged at 4000 g to get the mitochondria-enriched fraction, which was then washed twice with isolation buffer.

Confocal imaging and image analysis

Confocal imaging was performed using the Zeiss LSM510-meta, LSM710/ConfoCor 3 and Nikon A1R+ Ti-E with N-SIM and FCS microscope systems. An Ar-ion laser (for excitation of GFP or Alexa Fluor 488 at 488 nm), a He-Ne laser (for excitation of RFP, Alexa Fluor 546 and 594 at 543 nm) and a He-Ne laser (for excitation of Alexa Fluor 633 and mitoTracker DeepRed at 633 nm) were used with a 63×1.4 NA oil immersion objective. Dichroic mirrors used were HFT 488/543 and NFT 545. Emission filters BP530-550, BP565-615 and LP650 were used for imaging GFP, RFP and Mitotracker Deep Red FM, respectively. mito-Rosella-, mitoGFP- and mitoRFP-transfected cells were imaged in CO2-independent medium, maintaining conditions of live-cell imaging as described previously (Mitra and Lippincott-Schwartz, 2010). Mitochondria were imaged taking z-stacks with a z-interval of 0.25 µm. 3D projection images were generated in ImageJ. z-projections of the same images were generated and used to quantify the mitochondrial distribution [defined as (mitochondrial area/whole cell area)×100]. Colocalization analyses between p62 and mitochondria were performed using ZEN software. 50–100 cells were analyzed from more than five independent experiments for quantitative analyses.

FRAP

HeLa cells expressing mitoRFP and GFP-tagged MGRN1 or MGRN1ΔR were imaged using live-cell imaging conditions. A fixed small region of interest (ROI; 5×10 µm) within each cell was photobleached with a 561-nm laser at 100% power for 10 s. Image acquisition was performed every 10 s for 5 min. The total fluorescence intensity of the cell and fluorescent intensity in the bleached region was quantified using ImageJ. The signal was corrected for overall photobleaching using signal from an unbleached cell in the same field of view as described previously (Mitra and Lippincott-Schwartz, 2010). Data was plotted using Excel (Microsoft). Raw data (without postprocessing) were used for the quantification. For representative images, brightness, contrast and cropping were performed with Photoshop (CS2; Adobe).

Measurement of changes in mitochondrial membrane potential using TMRE

HeLa cells were loaded with 200 nM TMRE for 20 min. A single image was taken focusing on a single cell and keeping the plate on the microscope stage, then 10 µM CCCP was added and the TMRE intensity was monitored for 10 min at 30-s intervals. The initial fluorescence intensity (F0) and the fluorescence intensity at time ‘t’ (Ft) were calculated using ImageJ. This was performed for eight cells per set taken from five independent experiments. The average Ft/F0 is plotted against time.

Determination of intracellular Ca2+

HeLa cells were treated with BAPTA-AM (75 μM, 6 h), CCCP (20 µM, 6 h) and the intracellular free Ca2+ concentration was measured using 10 µM fura-2-AM in Tyrodes solution (10 mM Hepes pH 7.4, 10 mM NaCl, 3 mM KCl, 2 mM CaCl2 and 10 mM glucose). Cell loading of fura-2 was carried out at 37°C for 30 min to enhance dye uptake. Ca2+ concentrations were measured as the ratio of fluorescence intensities obtained when samples were excited at 340 nm and 380 nm sequentially. Rmax and Rmin were calculated as previously described (Pérez et al., 1998; Grynkyewicz et al., 1985). An apparent Kd for Fura-2-Ca was taken as 224 nM.

Brain lysates from mice

Transgenic mice expressing HuPrP(A117V) have been generated and characterized previously (Hegde et al., 1998; Rane et al., 2010). Frozen brain lysates from non-transgenic and transgenic mice were used for analyses. For this brains from mice killed (in compliance with the guidelines specified by the National Institutes of Health) had been weighed and dissolved in 9 volumes of 1% SDS and 0.1 M Tris-HCl, pH 8.0 by passing through a 16 gauge needle (twenty times) followed by 20 gauge needle (twenty times). Equivalent amounts of lysates were analysed by immunoblotting.

We thank S. Banerjee, I. R. Nabi, H.-S. Cho, R. Mattera, K.-L. Lim, T. Ohta, R. Devenish and E. Snapp for plasmids; D. Mukhopadhyay; M. M. Panicker and R. S. Hegde for cells and antibodies; P. K. Chakraborty at Towa Optics (I) Pvt. Ltd. for help with microscopy experiments; members of the O.C. laboratory (P. Majumdar, D. Srivastava, Z. Kaul and D. Mookherjee) for their help and support throughout the study.

Author contributions

O.C. and R.M. conceived the project and designed the experiments. R.M. performed most of the experiments with contributions from O.C. R.M. and O.C. interpreted the results and wrote the paper.

Funding

This work was supported by the ‘Integrative Biology on Omics Platform Project’, intramural funding of the Department of Atomic Energy (DAE), Government of India.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information