Vladimir V. Lupashin

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. “Deep etch” EM of purified COG revealed an ∼37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.

Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.

The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins.

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.

COG Complex‐Mediated Recycling of Golgi Glycosyltransferases is Essential for Normal Protein Glycosylation

Defects in conserved oligomeric Golgi (COG) complex result in multiple deficiencies in protein glycosylation. On the other hand, acute knock‐down (KD) of Cog3p (COG3 KD) causes accumulation of intra‐Golgi COG complex‐dependent (CCD) vesicles. Here, we analyzed cellular phenotypes at different stages of COG3 KD to uncover the molecular link between COG function and glycosylation disorders. For the first time, we demonstrated that medial‐Golgi enzymes are transiently relocated into CCD vesicles in COG3 KD cells. As a result, Golgi modifications of both plasma membrane (CD44) and lysosomal (Lamp2) glycoproteins are distorted. Localization of these proteins is not altered, indicating that the COG complex is not required for anterograde trafficking and accurate sorting. COG7 KD and double COG3/COG7 KD caused similar defects with respect to both Golgi traffic and glycosylation, suggesting that the entire COG complex orchestrates recycling of medial‐Golgi‐resident proteins. COG complex‐dependent docking of isolated CCD vesicles was reconstituted in vitro, supporting their role as functional trafficking intermediates. Altogether, the data suggest that constantly cycling medial‐Golgi enzymes are transported from distal compartments in CCD vesicles. Dysfunction of COG complex leads to separation of glycosyltransferases from anterograde cargo molecules passing along secretory pathway, thus affecting normal protein glycosylation.

Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes.

Quantitative Proteomic and Genetic Analyses of the Schizophrenia Susceptibility Factor Dysbindin Identify Novel Roles of the Biogenesis of Lysosome-Related Organelles Complex 1

The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture (SILAC) quantitative proteomics combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex, many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1–2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.

Role of vesicle tethering factors in the ER–Golgi membrane traffic

Tethers are a diverse group of loosely related proteins and protein complexes grouped into three families based on structural and functional similarities. A well‐accepted role for tethering factors is the initial attachment of transport carriers to acceptor membranes prior to fusion. However, accumulating evidence indicates that tethers are more than static bridges. Tethers have been shown to interact with components of the fusion machinery and with components involved in vesicle formation. Tethers belonging to the three families act at the same stage of traffic, suggesting that they mediate distinct events during vesicle tethering. Thus, multiple tether‐facilitated events are required to provide selectivity to vesicle fusion. In this review, we highlight findings that support this model.

Cog1p Plays a Central Role in the Organization of the Yeast Conserved Oligomeric Golgi Complex

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved peripheral membrane oligomeric protein complex that is involved in intra-Golgi protein trafficking. The COG complex is composed of eight subunits that are located in two lobes; Lobe A contains COG1–4, and Lobe B is composed of COG5–8. Both in vivo and in vitro protein-protein interaction techniques were applied to characterize interactions between individual COG subunits. In vitro assays revealed binary interactions between Cog2p and Cog3p, Cog2p and Cog4p, and Cog6p and Cog8p and a strong interaction between Cog5p and Cog7p. The two-hybrid assay confirmed these findings and revealed that Cog1p interacted with subunits from both lobes of the complex. Antibodies to COG subunits were utilized to determine the protein levels and membrane association of COG subunits in yeast Δcog1–8 mutants. As a result, we created a model of the protein-protein interactions within the yeast COG complex and proposed that Cog1p is a bridging subunit between the two COG lobes. In support of this hypothesis, we have demonstrated that Cog1p is required for the stable association between two COG subcomplexes.

Role of the conserved oligomeric Golgi (COG) complex in protein glycosylation

The Golgi apparatus is a central hub for both protein and lipid trafficking/sorting and is also a major site for glycosylation in the cell. This organelle employs a cohort of peripheral membrane proteins and protein complexes to keep its structural and functional organization. The conserved oligomeric Golgi (COG) complex is an evolutionary conserved peripheral membrane protein complex that is proposed to act as a retrograde vesicle tethering factor in intra-Golgi trafficking. The COG protein complex consists of eight subunits, distributed in two lobes, Lobe A (Cog1-4) and Lobe B (Cog5-8). Malfunctions in the COG complex have a significant impact on processes such as protein sorting, glycosylation, and Golgi integrity. A deletion of Lobe A COG subunits in yeasts causes severe growth defects while mutations in COG1, COG7, and COG8 in humans cause novel types of congenital disorders of glycosylation. These pathologies involve a change in structural Golgi phenotype and function. Recent results indicate that down-regulation of COG function results in the resident Golgi glycosyltransferases/glycosidases to be mislocalized or degraded.

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.