Irina D. Pokrovskaya

The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P < 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 ± 10% (P < 0.05) and FAS expression increased by 63 ± 8% (P < 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity.

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery.

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.

COG complexes form spatial landmarks for distinct SNARE complexes

Vesicular tethers and SNAREs are two key protein components of the intracellular membrane trafficking machinery. The COG (conserved oligomeric Golgi) complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Here, using yeast two hybrid and co-immunoprecipitation approaches, we show that three COG subunits, namely COG4, 6, and 8, are capable of interacting with defined Golgi SNAREs, namely STX5, STX6, STX16, GS27, and SNAP29. Comparative analysis of COG8-STX16 and COG4-STX5 interactions by a COG-based mitochondrial re-localization assay reveals that the COG8 and COG4 proteins initiate the formation of two different tethering platforms that can facilitate the redirection of two populations of Golgi transport intermediates to the mitochondrial vicinity. Our results uncover a role for COG subcomplexes in defining the specificity of vesicular sorting within the Golgi.

COG6 Interacts with a Subset of the Golgi SNAREs and Is Important for the Golgi Complex Integrity

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra‐Golgi vesicles. Using yeast two‐hybrid and co‐immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE‐binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE‐binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6–SNARE interactions are important for both COG6 localization and Golgi integrity.

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane‐associated protein complex that functions in a retrograde intra‐Golgi trafficking through associations with coiled‐coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex‐interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15‐positive COG complex‐dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP‐tagged and endogenous COG complex subunits associated with inclusions in a serovar‐independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v‐SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid‐cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi‐derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.

COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits.

The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole.

STEM tomography reveals that the canalicular system and α-granules remain separate compartments during early secretion stages in blood platelets.

Essentials How platelets organize their α‐granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis.

Multipronged interaction of the COG complex with intracellular membranes

The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.

Cog5–Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex

Significance In all eukaryotes, the docking and fusion of the vesicles that mediate intracellular trafficking requires multisubunit tethering complexes (MTCs). MTCs are thought to mediate the initial interaction between the vesicle and its target membrane and to orchestrate the assembly of the protein fusion machinery. The largest family of MTCs—of which the conserved oligomeric Golgi (COG) complex is a well-studied member—has been recalcitrant to structural characterization, presumably owing to the size and intrinsic flexibility of the complexes and their constituent subunits. Here we report the initial characterization of subunit interactions within the COG complex by X-ray crystallography. Mutations in the conserved intersubunit interface may be responsible for human congenital glycosylation disorders. The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1–4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG’s eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5–Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5–Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.

COG lobe B sub-complex engages v-SNARE GS15 and functions via regulated interaction with lobe A sub-complex

The conserved oligomeric Golgi (COG) complex is a peripheral membrane protein complex which orchestrates tethering of intra-Golgi vesicles. We found that COG1-4 (lobe A) and 5–8 (lobe B) protein assemblies are present as independent sub-complexes on cell membranes. Super-resolution microscopy demonstrates that COG sub-complexes are spatially separated on the Golgi with lobe A preferential localization on Golgi stacks and the presence of lobe B on vesicle-like structures, where it physically interacts with v-SNARE GS15. The localization and specific interaction of the COG sub-complexes with the components of vesicle tethering/fusion machinery suggests their different roles in the vesicle tethering cycle. We propose and test a novel model that employs association/disassociation of COG sub-complexes as a mechanism that directs vesicle tethering at Golgi membranes. We demonstrate that defective COG assembly or restriction of tethering complex disassembly by a covalent COG1-COG8 linkage is inhibitory to COG complex activity, supporting the model.