ABSTRACT The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI. This article has an associated First Person interview with the first author of the paper.

ABSTRACT The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field.

ABSTRACT The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT–PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT–PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT–PIX complex in health and disease.

Summary ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization, instead of the six in human, enabling a more precise functional analysis. Genetic invalidation of acapA resulted in multinucleated cells with cytokinesis defects. Mutant acapA − cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A–GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that the ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA•GTP mutant in wild-type cells led to a significant reduction in filopodia length. Together, our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism.

The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are ~20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN.

Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. GIT1/p95-APP1 is a member of a family of GTPase-activating proteins for ARF GTPases that affect endocytosis, adhesion and migration. GIT1 associates with paxillin and a complex including the Rac/Cdc42 exchanging factors PIX/Cool and the kinase PAK. In this study, we show that overexpression of βPIX induces the accumulation of endogenous and overexpressed GIT1 at large structures similar to those induced by an ArfGAP-defective mutant of GIT1 (p95-C2). Immunohistochemical analysis and immunoelectron microscopy reveal that these structures include the endogenous transferrin receptor. Time-lapse analysis during motogenic stimuli shows that the formation and perinuclear accumulation of the p95-C2-positive structures is paralleled by inhibition of lamellipodium formation and cell retraction. Both dimerization and a functional SH3 domain of βPIX are required for the accumulation of GIT1 in fibroblasts, which is prevented by the monomeric PIX-PG-ΔLZ. This mutant also prevents the formation of endocytic aggregates and inhibition of neurite outgrowth in retinal neurons expressing p95-C2. Our results indicate that βPIX is an important regulator of the subcellular distribution of GIT1, and suggest that alteration in the level of expression of the complex affects the endocytic compartment and cell motility.

ADP-ribosylation factors (Arfs), key regulators of intracellular membrane traffic, are known to exert multiple roles in vesicular transport. We previously isolated eight temperature-sensitive (ts) mutants of the yeast ARF1 gene, which showed allele-specific defects in protein transport, and classified them into three groups of intragenic complementation. In this study, we show that the overexpression of Glo3p, one of the GTPase-activating proteins of Arf1p (ArfGAP), suppresses the ts growth of a particular group of the arf1 mutants ( arf1-16 and arf1-17 ). Other ArfGAPs do not show such a suppression activity. All these ArfGAPs show sequence similarity in the ArfGAP catalytic domain, but are divergent in the rest of molecules. By domain swapping analysis of Glo3p and another ArfGAP, Gcs1p, we have shown that the non-catalytic C-terminal region of Glo3p is required for the suppression of the growth defect in the arf1 ts mutants. Interestingly, Glo3p and its homologues from other eukaryotes harbor a well-conserved repeated ISSxxxFG sequence near the C-terminus, which is not found in Gcs1p and its homologues. We name this region the Glo3 motif and present evidence that the motif is required for the function of Glo3p in vivo.

Phosphoinositide (PI) 3-kinase enhancer (PIKE) is a brain-specific GTPase that binds to PI 3-kinase and stimulates its lipid kinase activity. It exists in two forms: the first to be identified, PIKE-S, is shorter and exclusively nuclear; by contrast, the longer form, PIKE-L, resides in multiple intracellular compartments. Nerve growth factor treatment leads to PIKE-S activation by triggering the nuclear translocation of phospholipase C (PLC)-γ1, which acts as a physiological guanine nucleotide exchange factor (GEF) for PIKE-S through its Src-homlogy 3 (SH3) domain. Cytoplasmic PI 3-kinase and its lipid product phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5) P 3 ] regulate the membrane translocation and activation of many signaling molecules by binding to their pleckstrin homology (PH) domains. However, little is known about the physiological roles of their nuclear counterparts. The nuclear PLC-γ1/PIKE-S/PI 3-kinase signaling pathway seems to be an extension of the crosstalk between cytoplasmic PLC-γ1 and PI 3-kinase. PIKE-L contains a C-terminal extension consisting of an ADP ribosylation-GTPase-activating protein (ArfGAP) domain and two ankyrin repeats in addition to the N-terminal GTPase domain. PIKE-L could have additional, extranuclear functions, including regulation of postsynaptic signaling by metabotropic glutamate receptors.