EXOMER COMPLEX

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Transport of proteins between membrane-bound compartments is a tightly regulated process involving vesicular membrane carriers. A classical membrane vesicle is created by membrane deformation achieved in part by cytosolic "coat" proteins. Coat proteins bind and shape membranes, and they also play an important role in selecting cargo incorporated into transport carriers. Recent studies have established the existence of a new membrane coat complex responsible for trafficking certain cargo proteins from the trans-Golgi network (TGN) to the plasma membrane in yeast. This coat, named "Exomer", is interesting as an alternative to the better-known TGN coat systems involving Clathrin and Adaptor Proteins. Exomer is also quite interesting as a model for regulated transport. Under steady-state conditions, cargo trafficked by exomer is localized in a stable internal compartment, but in response to stress or progression through the cell-cycle cargo is rapidly mobilized to the plasma membrane in a polarized manner. The proper localization of two Exomer cargo proteins in particular (Chs3 and Fus1) is easily monitored by simple assays, making this an excellent system for in-depth mechanistic studies. We are pursuing structural and biochemical studies of Exomer in order to understand how it functions. We currently have diffracting crystals of a fully functional exomer complex, having collected a native 2.8A dataset on the CHESS A1 beamlime. We are unable to grow crystals of the SeMet-substituted protein, so we are now resorting to heavy-atom soaks for MIR/MIRAS/SIRAS. We are unable to screen for derivatives on a home source because our crystals do not diffract strongly enough using home-source X-rays. An insertion device beamline is ideal, as the best we can do is 2.8A at CHESS A1. We also would like to take advantage of tunable wavelength for attempts at MIRAS/SIRAS.