Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. Here we report the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila melanogaster Sec15. This C-terminal domain was found to bind a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. We also provide evidence that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-Å crystal structure of the C-terminal domain revealed a novel fold consisting of ten α-helices equally distributed between two subdomains (N and C subdomains). We show that the C subdomain, mainly via a single helix, is sufficient for Rab binding.
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We thank the staff at Lawrence Berkeley National Laboratory beamline 8.2.1 for their help with the X-ray data collection. We thank J. He and J.L. Fallon for their technical assistance and R. Cohen (University of Kansas, Lawrence, Kansas, USA) for the Rab11 antibody, Rab11-GFP line and Rab11 mutants. S.W. was supported in part by a training fellowship from the W.M. Keck Foundation to the Gulf Coast Consortia through the Keck Center for Computational and Structural Biology and by a grant from the Welch Foundation (Q-0581) to F.A.Q. S.Q.M. was supported by the US National Institutes of Health grant EY07001 and is a member of the Medical Scientist Training Program. A significant part of the work in the laboratories of F.A.Q. and H.J.B. was supported by the Howard Hughes Medical Institute. Work in F.P.'s laboratory was supported by the Medical Research Council and the Biotechnology and Biological Sciences Research Council.
The authors declare no competing financial interests.
Sequence alignment of the predicted Sec15 domain (PDF 347 kb)
Electrostatic surface properties of the C-terminal domain and an unusual interaction of Arg572. (PDF 593 kb)
Model of the interaction between the C-subdomain and Rab11. (PDF 151 kb)
Interaction of the C-subdomain mutants with Rab11 (PDF 85 kb)
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