STRUCTURE DETERMINATION OF THE SAR/SEC23/PEPTIDE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is the third in a series of proposals that aims at obtaining structural information on the yeast COPII vesicular coat. COPII is a 420 kDa pentameric complex responsible for budding transport vesicles from the endoplasmic reticulum (ER) and delivering the specific cargo contents to the Golgi complex. In the first proposal we focused on the inner shell of the COPII coat, the Sec23/24-Sar1 complex. We were able to solve this 210 kDa structure by MAD and molecular replacement from data collected at X-25 and at CHESS (manuscript published). The second proposal focused on the selectivity of the COPII coat for cargo and for ER-to-Golgi SNARE fusogenic proteins. We were able to determine the structures of three complexes of Sec24 bound to cargo and SNARE sequences (manuscript submitted). In the current proposal we focus on the outer shell of COPII, comprising the Sec13/31 complex. Sec13/31 is functionally (but not structurally) homologous to clathrin-it polymerizes the COPII inner shell on the ER membrane and causes membrane deformation. Since, like clathrin, Sec13/31 is a long, flexible structure capable of oligomerizing (as seen in EM images) it is unlikely that the intact complex can be crystallized. Therefore, we have dissected the protein into what we believe are the two functionally-interesting fragments, and these constitute the two projects that we propose in this beamline application.