Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150Glued subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo β-1,4-galactosyltransferase (Galtase)-YFP but not the COPI-dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway.

Scy1-like 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, was recently identified as the gene product altered in muscle-deficient mice, which suffer from motor neuron degeneration and cerebellar atrophy. To determine the function of Scyl1, we have now used a mass spectrometry-based screen to search for Scyl1-binding partners and identified components of coatomer I (COPI) coats. The interaction was confirmed in pull-down assays, and Scyl1 co-immunoprecipitates with ␤COP from brain lysates. Interestingly, and unique for a non-transmembrane domain protein, Scyl1 binds COPI coats using a C-terminal RKLD-COO ؊ sequence, similar to the KKXX-COO ؊ COPI-binding motif found in transmembrane endoplasmic reticulum (ER) proteins. Scyl1 co-localizes with ␤COP and is localized, in an Arf1-independent manner, to the ER-Golgi intermediate compartment and the cis-Golgi, sites of COPI-mediated membrane budding. The localization and binding properties of Scyl1 strongly suggest a function in COPI transport, and inhibitory RNA-mediated knock down of the protein disrupts COPI-mediated retrograde traffic of the KDEL receptor to the ER without affecting anterograde traffic from the ER. Our data demonstrate a function for Scyl1 as an accessory factor in COPI trafficking and suggest for the first time that alterations in the COPI pathway result in neurodegenerative disease.The murine autosomal recessive neurodegenerative disease model "muscle-deficient" (mdf) displays clinical and histological features indicative of a progressive motor neuropathy (1). Homozygotes develop a posterior waddle at 4 -8 weeks of age followed by hind limb paralysis and forelimb weakness (1). Skeletal muscles exhibit a neurogenic atrophy, and there is progressive neurodegeneration of lower motor neurons (1, 2). Among the murine neuromuscular atrophies, the motor neuron degeneration in mdf is most similar to that seen in wobbler (1). Interestingly, mdf also suffers from symptoms indicative of cerebellar involvement including gait ataxia, abnormal hind limb posture, and tremor (3). Therefore, mdf appears to be linked to both neuromuscular atrophy and spinocerebellar ataxia and is thus an animal model for neuromuscular disease with cerebellar involvement in humans.Most recently, null mutations in the Scy1-like 1 (Scyl1) gene were determined to be responsible for the pathology seen in the mdf mouse (3). Scyl1 was initially identified as a ubiquitously expressed member of the Scy1-like family of protein kinases (4) and is a distant homologue of Scy1-like 2/CVAK104 (coated vesicle-associated kinase of 104 kDa), which is involved in the trafficking of clathrin-coated vesicles (CCVs) 3 (5-7). Scy1-like kinases are thought to be kinase-inactive as they lack several highly conserved residues essential for catalytic activity (8, 9). Scyl1 was indirectly implicated in clathrin-mediated endocytosis when it was identified as an in vitro binding partner for the ␣-and ␤2-ear domains of the clathrin adaptor protein-2 (AP-2) (in this study, ...

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf ⅐ GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf ⅐ GTP, rather than the formation of an Arf ⅐ GDP ⅐ BFA ⅐ GBF1 complex.

Localization of resident Golgi proteins to earlier ( cis) or later ( trans) Golgi compartments has traditionally required quantitative immunocytochemistry and electron microscopy, which are inaccessible to many researchers. For this reason, light microscopy has often been used, initially for localization of Golgi glycotransferases and, more recently, for other Golgi proteins (e.g., Arf1, GBF1, Rab6). Quantitation of light microscopic intra-Golgi localization can be problematic. We describe here a novel quantitative light microscopic methodology using linescans crossing the Golgi ribbon. Our method determines a localization for the unknown protein in a one-dimensional coordinate system in which 0.0 corresponds to localization of a cis marker and 1.0 to localization of a trans marker. We also describe a variant of this methodology in which Golgi morphology is simplified by nocodazole-induced dispersal into ministacks, allowing a fully automated analysis. In our assay, β1,4-galactosyltransferase-YFP and Golgin97 localize similarly to trans markers, whereas p115, GBF1, and p58-YFP are similarly near other cis markers. The medial Golgi protein α1,3–1,6-mannosidase II gives an intermediate localization in this assay. These methodologies may prove useful in instances where electron microscopy is technically difficult as well as when rapid analysis of large numbers of samples is required.

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.