The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles

Sivaram, Mylavarapu V. S.; Furgason, Melonnie Lynn Marie; Brewer, Daniel N.; Munson, Mary

doi:10.1038/nsmb1096

Cited by 93 publications

(125 citation statements)

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“…4A). The striking resemblance between Cog4-(525-785) and the known exocyst and Dsl1p structures, particularly those of Sec6p (residues 411-805) (18) and Tip20p (20), provides strong confirmation that at least some of the subunits composing these complexes are descended from a common progenitor. We therefore adopt the domain nomenclature initially proposed by Dong et al (15) and later In the left-hand pair of images, the surface is color-coded by sequence conservation (darker blue is more conserved).…”

Section: Structural Homology Between Cog4 and Other Tethering Complexmentioning

confidence: 77%

“…Subunits of the COG, exocyst, Dsl1p, and Golgiassociated retrograde protein (GARP) complexes share limited sequence homology (7,13) that has been suggested to reflect convergent evolution (13). Structures, most of them partial, have been reported for one COG complex subunit, two Dsl1p complex subunits, and four exocyst subunits (14)(15)(16)(17)(18)(19)(20); these structures contain one (Cog2p), two (Dsl1p, Sec15, Exo84p), three (Sec6p), four (Exo70p), or five (Tip20p) helix bundle domains. In each of the exocyst and Dsl1p complex structures, the tandem helical domains form an extended, rod-like array (20,21).…”

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confidence: 99%

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Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene

Richardson

Smith

Ungár

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The proper glycosylation of proteins trafficking through the Golgi apparatus depends upon the conserved oligomeric Golgi (COG) complex. Defects in COG can cause fatal congenital disorders of glycosylation (CDGs) in humans. The recent discovery of a form of CDG, caused in part by a COG4 missense mutation changing Arg 729 to Trp, prompted us to determine the 1.9 Å crystal structure of a Cog4 C-terminal fragment. Arg 729 is found to occupy a key position at the center of a salt bridge network, thereby stabilizing Cog4's small C-terminal domain. Studies in HeLa cells reveal that this C-terminal domain, while not needed for the incorporation of Cog4 into COG complexes, is essential for the proper glycosylation of cell surface proteins. We also find that Cog4 bears a strong structural resemblance to exocyst and Dsl1p complex subunits. These complexes and others have been proposed to function by mediating the initial tethering between transport vesicles and their membrane targets; the emerging structural similarities provide strong evidence of a common evolutionary origin and may reflect shared mechanisms of action.congenital disorder of glycosylation ͉ Golgi apparatus ͉ multi-subunit tethering complex ͉ vesicle trafficking ͉ X-ray crystallography M ultiple lines of evidence implicate the conserved oligomeric Golgi (COG) complex in retrograde transport within the Golgi apparatus (1, 2). For example, partial knockdown of mammalian COG3 permitted exocytosis, while simultaneously blocking the retrograde transport of Shiga toxin from endosomes to the ER (3). COG was initially isolated from bovine brain on the basis of its ability to stimulate an in vitro Golgi transport assay (4). Two of its 8 subunits, Cog1/ldlB and Cog2/ ldlC, had previously been identified in a genetic screen for compromised cell surface receptor stability (5, 6). In the yeast Saccharomyces cerevisiae, the COG complex was discovered through studies of one of its subunits, Cog8p/Dor1p. Implicated in vesicle targeting to the Golgi apparatus, Cog8p was found to co-immunoprecipitate with 7 other polypeptides (7). The 7 associated polypeptides included two (Cog2p/Sec35p and Cog3p/ Sec34p) that had earlier been identified in genetic screens for temperature-sensitive mutations causing secretion defects (8). In further support of a role in trafficking to and within the Golgi, COG interacts physically with a large number of Golgi-localized trafficking factors, including (in yeast) the Rab family G protein Ypt1p, the SNAREs Sed5p, Ykt6p, Gos1p, and Sec22p, and COPI vesicle coat proteins (9).COG is one of several large protein complexes proposed to function as multisubunit tethering factors, mediating the initial interaction between transport vesicles and their target membranes (2, 10-12). For many of these complexes, a dearth of structural information has slowed the development of mechanistic models and has made it difficult to know how-or whether-to apply lessons learned from studies of one complex to another. Subunits of the COG, exocyst, Dsl1p, and Golgiassoci...

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Section: Structural Homology Between Cog4 and Other Tethering Complexmentioning

confidence: 77%

mentioning

confidence: 99%

Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene

Richardson

Smith

Ungár

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The X-ray structure revealed that Cog5 adopts a classic α-helical CATCHR fold previously observed in other COG subunits (Cog2, Cog4) and in the Dsl1 (Dsl1, Tip20), exocyst (Sec6, Sec15, Exo70, and Exo84), and GARP (Vps53, Vps54) complexes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Munc13, a protein implicated in synaptic vesicle docking and fusion, also displays the CATCHR fold (40).…”

Section: Discussionmentioning

confidence: 99%

“…A survey of the Protein Data Bank (PDB) using the Dali server (29) revealed strong structural homology with domains A and B of the exocyst subunit Exo70 (Z = 10.2) and somewhat weaker but significant structural homology with other CATCHR-family MTC subunits, including the exocyst subunits Exo84 and Sec6, the GARP complex subunit Vps54, the Dsl1 complex subunits Tip20 and Dsl1, and Cog4 (Z scores ranged from 4.4 to 7.6). Thus, Cog5 joins the growing list of MTC subunits that display the CATCHR fold and are apparentlydespite very low levels of sequence homology-derived from a single evolutionary progenitor (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

Section: Significancementioning

confidence: 99%

“…Four of the known MTCs-termed complexes associated with tethering containing helical rods (CATCHR) or quatrefoil complexes (2, 5)-contain subunits whose shared 3D structure implies a single evolutionary progenitor (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). These CATCHR-family MTCs include the Dsl1, Golgi-associated retrograde protein (GARP), exocyst, and conserved oligomeric Golgi (COG) complexes, and they contain three, four, eight, and eight subunits, respectively.…”

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confidence: 99%

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

Pokrovskaya

Climer

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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.I n eukaryotes, the transport of proteins and lipids among intracellular compartments is mediated by vesicular and tubular carriers under the direction of an elaborate protein machinery (1). Among the most complex and least well-characterized components of this machinery are the multisubunit tethering complexes (MTCs) (2). MTCs are thought to mediate the initial attachment (or tethering) between a trafficking vesicle and its target membrane through a constellation of interactions (3, 4). These may include binding of the MTC to activated Rab GTPases, coiled-coil proteins such as Golgins, vesicle coat proteins, SNAREs, Sec1/ Munc18 (SM) proteins, and/or membrane lipids. Elucidating the 3D structures of MTCs represents an important step toward a better understanding of their molecular functions.Four of the known MTCs-termed complexes associated with tethering containing helical rods (CATCHR) or quatrefoil complexes (2, 5)-contain subunits whose shared 3D structure implies a single evolutionary progenitor (6-16). These CATCHR-family MTCs include the Dsl1, Golgi-associated retrograde protein (GARP), exocyst, and conserved oligomeric Golgi (COG) complexes, and they contain three, four, eight, and eight subunits, respectively. Although X-ray or NMR structures have been reported for 14 of these 23 subunits, only one of the structures contains the full-length polypeptide (14). Perhaps more critically, only two subunit interactions-both wit...

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Integrative structure and function of the yeast exocyst complex

et al. 2020

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Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at~16 Å resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution Abbreviations: CATCHR, complexes associated with tethering containing helical rods; CXMS, chemical-crosslinking mass spectrometry; DSS, disuccinimidyl suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EM, electron microscopy; GMM, Gaussian mixture model; IMP, integrative modeling platform; LC/MS, liquid chromatography and mass spectrometry; MTC, multisubunit tethering complex; PH domain, Pleckstrin homology; PMI, python modeling interface; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation; SNARE, soluble NSF attachment protein receptor. Sai J. Ganesan and Michael J. Feyder authors contributed equally to this work.

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The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles

Cited by 93 publications

References 14 publications

Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene

Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene

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

Integrative structure and function of the yeast exocyst complex

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