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...

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

ABSTRACTFusion of intracellular trafficking vesicles is mediated by the assembly of soluble N-ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. Here, we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its three-helical Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-d 5), as Trp48-d 5 in azurin, and as the photogenerated neutral tryptophan radical, Trp48-d 5 •, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp-d 5 bands relative to l-Trp-h 5 enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-h 5. The Raman intensities vary between l-Trp-d 5 and l-Trp-h 5; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-d 5 •, was generated in azurin with a slightly lower radical quantum yield than for Trp48-h 5 •. The visible resonance Raman spectrum of Trp48-d 5 • is different from that of Trp48-h 5 •, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp-d 5 is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-h 5.

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