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The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles Mylavarapu V S Sivaram, Melonnie L M Furgason, Daniel N Brewer & Mary Munson The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite o10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins. In eukaryotic cells, trafficking of protein and lipid cargo through the secretory pathway is mediated by membrane-bound vesicles1. Fusion of vesicles to the correct target membrane is achieved by the concerted action of tethering complexes and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs). The exocyst is a conserved octameric tethering complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) required for polarized secretion at the plasma membrane2,3. The Sec6p subunit from Saccharomyces cerevisiae is a helical dimer that binds the plasma membrane SNARE Sec9p and may regulate exocytic SNARE complex assembly4. Purified recombinant Sec6p directly binds the exocyst subunits Exo70p, Sec10p and Sec8p (Supplementary Fig. 1 online), consistent with previous biochemical and yeast two-hybrid studies5–8. Sec6p contains an independently folded, monomeric C-terminal domain (Sec6CT2; residues 411–805)4 that is essential for Sec6p function in vivo. Here, we present the structure of Sec6CT2 at 2.4-A˚ resolution and show that it is sufficient for interaction with both Exo70p and Sec10p, but not with Sec8p (Fig. 1 and Supplementary Table 1, Supplementary Methods and Supplementary Figs. 1–3 online). Sec6CT2 consists of three tandem helical-bundle subdomains, which contain five, four and three helices each, connected by long, solvent-exposed loops and short turns (Fig. 1). Each subdomain has a distinct hydrophobic core, and the overall structure is stabilized by interhelical interactions between the subdomains. Many of these core and interhelical residues are highly conserved across Sec6 orthologs (Supplementary Fig. 4 online). Structure-based sequence alignment also reveals a number of highly conserved amino acids on the surface of Sec6CT2, suggesting sites of conserved protein-protein interactions.
The importance of the Sec6CT2 domain in exocyst function is highlighted by the yeast sec6-4 temperature-sensitive mutant, L633P9. Leu633 packs in the hydrophobic core of subdomain B (Fig. 1), and its mutation to proline probably destabilizes helix a6 and disrupts hydrophobic packing contacts. The reversibility of the temperaturesensitive phenotype in the absence of new protein synthesis10 suggests that the nonpermissive temperature may cause only a local, reversible destabilization. The overall fold of the first two bundles of Sec6CT2 is remarkably similar to three other exocyst structures recently reported, namely the Sec15 C-terminal domain (Sec15CT; two bundles) from Drosophila melanogaster11, the yeast Exo84p C-terminal domain (Exo84CT; two bundles)8 and the nearly full-length Exo70p from yeast (four bundles, named A–D)8,12. Although these proteins have o10% sequence identity with Sec6p, they fold into the same novel structural motif, comprised of tandem helical bundles with comparable dimensions along the long axis (B80 A˚ for two bundles; Fig. 2a). The most notable similarity among these structures is their helical topology. Each structure is composed of at least two right-handed mixed antiparallel/parallel helical bundles packed together in an end-on arrangement (schematic diagram in Fig. 2b). All the helices on one side of the bundles are parallel in the direction of the top of the bundle. The helices on the other side, in contrast, are all parallel facing the bottom. The identical helical-bundle topology in all four exocyst
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Figure 1 Structure of the Sec6p C-terminal domain. (a) Ribbon backbone trace, right side view. (b) Left side view; rotated B1801 about the vertical axis with respect to a. The N and C termini, the N-terminal tag and the secondary structural elements are labeled. The position of the sec6-4 temperature-sensitive mutation (Leu633) is shown as a brown space-filled side chain.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA. Correspondence should be addressed to M.M. (
[email protected]). Received 12 December 2005; accepted 21 April 2006; published online 14 May 2006; doi:10.1038/nsmb1096
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B R I E F C O M M U N I C AT I O N S electron micrographs of purified mammalian complexes13. Comparable helical-bundle structures may also exist in tethering complexes that regulate other steps in intracellular trafficking. Regions of sequence similarity between various exocyst subunits and members of the COG and GARP tethering complexes have been noted14, and these subunits also have high helical propensities. The structures of Sec6CT2, Exo70p, Exo84CT and Sec15CT define a conserved architecture for the exocyst complex and provide a basis for testing the biological significance of its protein-protein interactions. Future high-resolution structures of exocyst subunits, combined with characterization of structure-based mutants and quantitative binding studies, will ultimately lead to elucidation of the assembly and disassembly mechanism of the exocyst complex and its role(s) in regulating polarized exocytosis.
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Figure 2 Sec6CT2 contains a similar helical-bundle motif to those of Exo70p, Exo84CT and Sec15CT. (a) Alignments of Sec6CT2 subdomains A and B (Sec6CT2AB) against the Exo70AB and Exo70CD helical bundles (PDB entry 2B1E), Exo84CT (PDB entry 2D2S) and Sec15CT (PDB entry 2A2F). The N terminus of each structure is at the bottom left. Pairwise comparisons between the Sec6CT bundles and individual bundles of the other exocyst proteins have main chain r.m.s. deviations between 3 and 4 A˚. (b) Topology of the helical-bundle motif.
subunits strongly suggests divergent evolution of the exocyst subunits from a common ancient ancestor. Although it is formally possible that the identical architecture arose through convergent evolution, this scenario is unlikely given the variety of topologies observed that form stable helical bundles. Although the overall helical-bundle topology is conserved among the exocyst structures, the surface features differ considerably. The shapes of the protein surfaces diverge owing to different bundle compositions (three to five helices of varying length and packing angles) and loop lengths. Moreover, the surface distributions of both charged and hydrophobic residues are not conserved (Supplementary Fig. 5 online). These changes result in each exocyst subunit presenting a unique molecular surface, creating individual specificities for protein-protein interactions. The remaining exocyst subunits, as well as the N-terminal domains of Sec6p, Sec15p and Exo84p, may also fold into similar helical-bundle structures, as they are predicted to be predominantly helical (Supplementary Fig. 6 online). The idea of a conserved exocyst architecture is reinforced by the size and shape similarity of the ‘arms’ observed in
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Accession code. Protein Data Bank: Coordinates have been deposited with accession code 2FJI. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGMENTS We are grateful to S. Ryder, W. Royer, W. Kobertz and R. Gilmore for critical reading of this manuscript and discussions. Thanks to the staff at the X25 and X29 beamlines at the National Synchrotron Light Source and to D. Lambright, B. van den Berg, S. Eathiraj and members of the Lambright laboratory for help with structure determination. This work was supported by US National Institutes of Health grant GM068803 to M.M. and an American Heart Association award to M.V.S.S. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. 2. 3. 4.
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