ABSTRACT Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13–Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only E3 proteins known to cooperate with Ubc13–Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. Here, we report a contribution of Ubc13–Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13–Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3 proteins for Ubc13–Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13–Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13–Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting. This article has an associated First Person interview with the first author of the paper .

Phosphatidylinositol 3-phosphate [PtdIns(3) P ] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3) P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3) P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4 C407A , the number of PtdIns(3) P -decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4 C407A , which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

The myotubularins are a large family of phosphoinositide-specific phosphatases with substrate specificity for PtdIns3 P and PtdIns(3,5) P 2 . In addition to an N-terminal PH-GRAM (PH-G) domain and a signature catalytic domain shared with other family members, MTMR3 contains a C-terminal FYVE domain. We show that the FYVE domain of MTMR3 is atypical in that it neither confers endosomal localisation nor binds to the lipid PtdIns3 P . Furthermore the FYVE domain is not required for in vitro enzyme activity of MTMR3. In contrast, the PH-GRAM domain is able to bind to phosphoinositide lipids, of which the allosteric regulator PtdIns5 P is the preferred partner. Consequently, generation of PtdIns5 P at the plasma membrane by ectopic expression of the bacterial phosphatase IpgD leads to a translocation of MTMR3 that requires the PH-G domain. Deletion of the PH-G domain leads to loss of activity of MTMR3 in vitro, and surprisingly, when combined with an active site mutation, accumulates the protein on the Golgi complex.

TOM1 and its related proteins, TOM1-like1 (TOM1-L1) and TOM1-like2 (TOM1-L2), constitute a subfamily of the VHS domain protein family. We have recently shown that endofin, a FYVE domain protein associated with the early endosome, is able to recruit cytosolic TOM1 onto endosomal membranes. To reveal the biological consequence of endofin-mediated endosomal recruitment of TOM1, we have identified the clathrin heavy chain as a major interacting protein for TOM1. Optimal clathrin binding by TOM1 involves three sites: residues 300-321, 321-326 and a putative clathrin-binding box at residues 362-366 ( 362 LEDEF 366 ). Although residues 321-326 could function independently as a weak clathrin-binding motif, deletion of amino acids 300-321 or mutation of 362 Leu and 364 Asp to Ala residues reduced the binding of clathrin to TOM1. A fragment lacking amino acids 300-322 and containing 362 Leu and 364 Asp to Ala mutations lost the ability to interact with clathrin. Remarkably, overexpression of endofin led to a massive and specific recruitment of clathrin [but not dynamin, or the adaptor protein (AP) complexes, AP1, AP2 or AP3] onto endofin-positive endosomes. Although SARA is homologous to endofin, it did not interact with the C-terminal region of TOM1. Examination of chimeric proteins of endofin and SARA suggests that the C-terminal half of endofin is responsible for interaction with the C-terminal region of TOM1 and for recruitment of TOM1 and clathrin to endosomes. The correlation between the ability of endofin to interact with the C-terminal domain of TOM1 and clathrin recruitment suggests that endofin may recruit clathrin via TOM1. Indeed, a chimeric protein consisting of TOM1 fused to two FYVE domains derived from endofin has the ability to recruit clathrin onto endosomal structures. Moreover, we show that affinity-purified TOM1 antibody can abolish binding of clathrin to the C-terminal region of TOM1. Upon microinjection into cells, this antibody reduced the membrane association of clathrin. These results, taken together, suggest that TOM1 is an important molecule for membrane recruitment of clathrin, and that endofin is able to exploit this recruitment at the endosome.

Phosphatidylinositol-3-phosphate [PtdIns(3) P ] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3) P in these processes, it is important to identify PtdIns(3) P -binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3) P in vitro and partially colocalizes with PtdIns(3) P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

Rab5, a member of the small GTPase family of proteins, is primarily localized on early endosomes and has been proposed to participate in the regulation of early endosome trafficking. It has been reported that phosphatidylinositol 3-kinases and FYVE domain proteins, such as EEA1, can be recruited onto early endosomes and act as Rab5 effectors. SARA (Smad anchor for receptor activation), also a FYVE domain protein, was initially isolated as a participant in signal transduction from the transforming growth factorβ receptor. Overexpressed SARA has been found on EEA1-positive early endosomes. In this report, we show that endogenous SARA is present on early endosomes and overexpression of SARA causes endosomal enlargement. Functionally, SARA overexpression significantly delays the recycling of transferrin. The transferrin receptor distributed on the cell surfaces was also greatly reduced in cells overexpressing SARA. However, the internalization rate of transferrin is not affected by SARA overexpression. The morphological and functional alterations caused by SARA overexpression resemble those caused by overexpression of Rab5:GTP mutant Rab5Q79L. Finally,all SARA-mediated phenotypic changes can be counteracted by overexpression Rab5:GDP mutant Rab5S34N. These results collectively suggested that SARA plays an important functional role downstream of Rab5-regulated endosomal trafficking.

FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.