Abstract
The conserved oligomeric Golgi (COG) complex is strongly implicated in retrograde vesicular trafficking within the Golgi apparatus. Although its mechanism of action is poorly understood, it has been proposed to function by mediating the initial physical contact between transport vesicles and their membrane targets. An analogous role in tethering vesicles has been suggested for at least six additional large multisubunit complexes, including the exocyst, a complex essential for trafficking to the plasma membrane. Here we report the solution structure of a large portion of yeast Cog2p, one of eight subunits composing the COG complex. The structure reveals a six-helix bundle with few conserved surface features but a general resemblance to recently determined crystal structures of four different exocyst subunits. This finding provides the first structural evidence that COG, like the exocyst and potentially other tethering complexes, is constructed from helical bundles. These structures may represent platforms for interaction with other trafficking proteins including SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) and Rabs.
Highlights
The conserved oligomeric Golgi (COG)3 complex is essential for normal Golgi morphology and function, yet its structure and precise role are poorly understood
Structures of several subunits of the exocyst complex have been determined (18 –23). These results are of particular relevance to COG because detectable sequence homology has been reported between regions of some exocyst and COG subunits (17, 24), the possibility that this homology represents convergent evolution has been raised recently (25)
The COG complex is a heterooctamer implicated in retrograde trafficking within the Golgi apparatus
Summary
Protein Overexpression and Purification—Recombinant Cog constructs were generated by PCR and expressed in E. coli BL21 as glutathione S-transferase fusion proteins using the expression plasmid pGEX-4T1 (GE Healthcare). Yeast cells were co-transformed with an excess of the resulting PCR product together with pSV15 that had been digested with BsgI and AvrII to remove sequences corresponding to Cog2p residues 57–262 and a portion of the 3Ј-flanking region. Data Analysis and Structure Calculation—Backbone and most side-chain resonances were assigned using gradient-enhanced HNCO, HNCACB, CBCA(CO)NH, (H)C(CO)NHTOCSY, HCCH-TOCSY, and 13C,1H HSQC-NOESY spectra and standard assignment procedures (35–37). H-bond restraints were assumed for regions of the protein exhibiting strongly helical chemical shift indices, 108 –128, 132–150, 159 –176, 185–207, 215–242, 249 –258. Structures calculated without these restraints were of similar energy and fold. Pairwise Z scores and root mean square deviations for each domain comparison were calculated using DaliLite (45)
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