Analysis Of The Transport Of Saccharomyces Cerevisiae Chitin Synthase 3 By The Exomer Secretory Vesicle Cargo Adaptor
A major feature of a eukaryotic cell is its ability to compartmentalize its functions by sequestering components into distinct membrane-bound organelles. Since membrane-embedded proteins cannot diffuse through the cell to travel between organelles, they must be sorted into transport vesicles, typically by coat complexes and their adaptors. The transmembrane protein Chitin Synthase 3 (Chs3) in Saccharomyces cerevisiae is an excellent model system to study some of these complex sorting and transport processes. Chs3 cycles between specific locations on the plasma membrane (PM) where it synthesizes chitin for the yeast cell wall, and retention in trans-Golgi network (TGN) compartments where it is inactive. Exomer, a novel protein complex found in fungi, acts as an adaptor complex for the transport of Chs3 and several other proteins from the TGN to the PM. The exomer complex is composed of the core subunit protein Chs5 and paralagous adaptor proteins known as ChAPs (Chs5-Arf1-binding Proteins), and is recruited to the membrane by the small GTPase Arf1. I have determined the minimal functional fragment of Chs5, which I have shown interacts with Arf1, likely contributing to exomer recruitment. The ChAPs are responsible for binding cargo, and Chs6 is required for transport of Chs3. Therefore, I examined Chs6 protein levels throughout the cell cycle and incorporation into complexes. When Chs6 levels were held constant by replacing its promoter with another, there was an effect on Chs3 transport only in one yeast background, indicating this regulation is only required under certain conditions. I also show that different segments of the Chs3 N-terminus mediate distinct trafficking steps. I present a crystal structure of residues 10-27 bound to the exomer complex, which are residues known to mediate retention and also seem to play a role in internalization. Residues 2852 are involved in transport to the plasma membrane and recycling out of the endosomes to prevent degradation. Together, these findings contribute to our understanding of how proteins are transported by exomer, and how cycling of a transmembrane protein can be regulated.
Cell Biology; Membrane trafficking; Yeast
Fromme, Joseph Chris
Emr, Scott David; Collins, Ruth N.
Molecular and Cell Biology
Ph. D., Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis