Mouse prenylated Rab acceptor is a novel Golgi membrane protein

We have cloned a mouse prenylated Rab acceptor (mPRA), which interacts with various Rab proteins in the yeast two-hybrid system. This study investigated its intracellular localization and characterized the localization signal. The mPRA was found to be an integral membrane protein that was localized to the Golgi complex at steady state as determined by confocal fluorescence microscopy. With green fluorescent protein attached to the N-terminus of mPRA, the fusion protein was expressed in BHK cells and was shown to exhibit the same Golgi localization as the native mPRA. Systematic truncations from the N- and C-termini of mPRA revealed that the entire N-terminal half (91 residues) of the protein was dispensable for the Golgi localization. In contrast, deletion of only 5 residues from the C-terminus diminished the Golgi localization of mPRA, leading to its accumulation in the ER. The data indicate that the C-terminal half (94 residues) of mPRA is necessary and sufficient for proper folding, ER export, and Golgi localization. The Golgi localization of mPRA suggests that it may play a role in the structural organization and function of the Golgi complex.     

Cohen syndrome-associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity

Loss-of-function mutations in the gene COH1, also known as VPS13B, lead to autosomal recessive Cohen syndrome. However, the cellular distribution and function of the encoded protein COH1 (3997 amino acids), which lacks functional homologies to other mammalian proteins, have remained enigmatic. We show here that COH1 is a peripheral Golgi membrane protein that strongly co-localizes with the cis-Golgi matrix protein GM130. Consistent with its subcellular localization, COH1 depletion using RNAi causes fragmentation of the Golgi ribbon into ministacks. Disruption of Golgi organization observed in fibroblasts from Cohen syndrome patients suggests that Golgi dysfunction contributes to Cohen syndrome pathology. In conclusion, our findings establish COH1 as a Golgi-associated matrix protein required for Golgi integrity.     

Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa.

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.     

Identification and characterization of a novel Golgi protein, golgin-67

In the course of screening a lambdagt11 human leukemic T-cell cDNA expression library with an antibody specific to the mitotic target of Src, Sam68, we identified and cloned a cDNA encoding a novel protein with a predicted molecular mass of 51.4 kDa. Polyclonal antibodies raised to a His(6)-tagged construct of this protein, detected a approximately 67-kDa protein in immunoprecipitation experiments, and cytological studies showed that this protein localized to the Golgi complex, through colocalization experiments with specific Golgi markers. Therefore, we designated this protein golgin-67. Sequence analysis revealed that golgin-67 is a highly coiled-coil protein, with potential Cdc2 and Src kinase phosphorylation motifs. It has sequence homologies to other Golgi proteins, including the coatamer complex I vesicle docking protein, GM130. Structurally, golgin-67 resembles, golgin-84, an integral membrane Golgi protein with an N-terminal coiled-coil domain and a single C-terminal transmembrane domain. The C-terminal region of golgin-67, which contains a predicted transmembrane domain, was demonstrated to be essential for its Golgi localization.     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

Identification of a novel protein (G210) specific to the Golgi apparatus

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.     

A novel type of regulation of the vimentin intermediate filament cytoskeleton by a Golgi protein

Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877-894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.     

A novel Golgi membrane protein is part of a GTPase-binding protein complex involved in vesicle targeting

Through two-hybrid interactions, protein affinity and localization studies, we previously identified Yip1p, an integral yeast Golgi membrane protein able to bind the Ras-like GTPases Ypt1p and Ypt31p in their GDP-bound conformation. In a further two-hybrid screen, we identified Yif1p as an interacting factor of Yip1p. We show that Yif1p is an evolutionarily conserved, essential 35.5 kDa transmembrane protein that forms a tight complex with Yip1p on Golgi membranes. The hydrophilic N-terminal half of Yif1p faces the cytosol, and according to two-hybrid analyses can interact with the transport GTPases Ypt1p, Ypt31p and Sec4p, but in contrast to Yip1p, this interaction is dispensable for Yif1 protein function. Loss of Yif1p function in conditional-lethal mutants results in a block of endoplasmic reticulum (ER)-to-Golgi protein transport and in an accumulation of ER membranes and 40-50 nm vesicles. Genetic analyses suggest that Yif1p acts downstream of Yip1p. It is inferred that Ypt GTPase binding to the Yip1p-Yif1p complex is essential for and precedes vesicle docking and fusion.     

A novel 58-kDa protein associates with the Golgi apparatus and microtubules

With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.     

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

The GOLD domain,a novel protein module involved in Golgi function and secretion

Background  

Members of the p24 (p24/gp25L/emp24/Erp) family of proteins have been shown to be critical components of the coated vesicles that are involved in the transportation of cargo molecules from the endoplasmic reticulum to the Golgi complex. The p24 proteins form hetero-oligomeric complexes and are believed to function as receptors for specific secretory cargo.     

Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen

We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142- 19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium- binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.     

Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.     

Signaling at the Golgi

The protein processing and trafficking function of the Golgi is intimately linked to multiple intracellular signaling pathways. Assembly of Golgi trafficking structures and lipid sorting at the Golgi complex is controlled and coordinated by specific phosphoinositide kinases and phosphatases. The intra-Golgi transport machinery is also regulated by kinases belonging to several functionally distinct families, for example, MAP kinase signaling is required for mitotic disassembly of the Golgi. However, the Golgi plays an additional, prominent role in compartmentalizing other signaling cascades that originate at the plasma membrane or at other organelles. This article summarizes recent advances in our understanding of the signaling network that converges at the Golgi.The Golgi apparatus is a dynamic structure that constantly exchanges proteins and lipids with other organelles. It is critical for organellar homeostasis that the different trafficking routes at the Golgi are precisely regulated. For example, the sorting and transport functions of the Golgi must be correctly coordinated with the overall activity of the secretory pathway. In addition, changes in Golgi structure and morphology are tightly controlled, which is particularly critical during mitosis, when the Golgi complex becomes disassembled for proper distribution between the dividing cells. It is therefore not surprising that diverse sets of signaling factors localize at the Golgi and control its function and shape.Phosphoinositide lipids have emerged as particularly important regulators of Golgi function. Reversible phosphorylation of the inositol headgroup of phosphatidylinositol creates seven distinct phosphoinositide species (Di Paolo and De Camilli 2006). These molecules serve as signal transducers at virtually every cellular membrane but have a particularly important role in controlling membrane traffic (Di Paolo and De Camilli 2006). A critical property of phosphoinositides is their tightly regulated spatial distribution. Recent studies have uncovered concentrated pools of these lipids at individual membranes including the Golgi (Roy and Levine 2004; De Matteis et al. 2005; Varnai and Balla 2008). Phosphoinositides often act in cooperation with small Ras-type GTPases and the interplay between phosphoinositides and GTPases from the ADP-ribosylation factor (Arf) and Ras-related in brain (Rab) families is essential for Golgi function (Behnia and Munro 2005; Mayinger 2009). How the lipid kinases and phosphatases that regulate Golgi phosphoinositides interact with other signaling pathway remains a challenging area of research.Whereas phosphoinositide signaling pathways are mainly controlled via extracellular signals that transmit metabolic status and growth conditions, Golgi function can also be regulated by signals that originate at other secretory organelles. Enhanced biosynthesis and processing of secretory proteins at the ER induces the activation of a signaling network that modulates intra-Golgi traffic and overall capacity of secretion (Sallese et al. 2009).Finally, there is mounting evidence that the Golgi serves as an important signaling platform for numerous signaling cascades that originate at the plasma membrane. The discovery that components of the Ras and the protein kinase A (PKA) pathways reside at the Golgi indicates that this organelle plays an important role in compartmentalizing signal transduction pathways (Quatela and Philips 2006; Sallese et al. 2009). This article will review our current understanding of signaling at the Golgi and also highlight the relevance of these processes for human disease.     

Re'COG'nition at the Golgi

The conserved oligomeric Golgi (COG) complex co-ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG-dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi-subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation.     

Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin

We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.