Identification and characterization of Vps74p, a coatomer and SNARE interacting protein involved in membrane traffic.

摘要

The ability to transport proteins and lipids through the secretory pathway is a fundamental process in all eukaryotic organisms. In humans defects in intracellular trafficking processes underlie many diseases highlighting the importance of maintaining the integrity protein sorting and membrane trafficking processes. It is generally accepted that the movement of proteins within the secretory pathway is mediated by protein-coated vesicles the formation of which couples protein sorting with transport specificity---processes that both specific and subject to regulatory control. The key players in transport vesicle formation are the vesicle coat proteins, protein cargo-sorting adapters, the SNARE proteins (which contribute to membrane fusion) and GTP-ases and their associated effector proteins.; In this thesis I established that some eukaryotic species contain only one member of the BET1/SFT1 Qc-SNARE family. Using functional complementation studies in yeast I showed that such genetic consolidation could be explained in part by single a SNARE taking on the functions of both Bet1 and Sft1---either by functional overlap or by direct functional substitution. I sought evidence of Bet1/Sft1 functional overlap in yeast and was able to demonstrate that over-expression of Bet1p could both suppress the temperature-sensitive growth defects in sft1-1 cells as well as support the growth of yeast cells in which the SFT1 gene had been deleted. These findings revealed an unexpected flexibility in membrane trafficking pathways between the Golgi and ER. I then identified proteins that contributed to this flexibility using genetics. I searched for proteins which when over-produced by would allow cells to grow in the absence of the otherwise essential Golgi trafficking protein, Sft1p. Using this gene dosage bypass suppressor screen I identified components of the COP1 vesicle formation machinery: coatomer subunits (beta-COP, gamma-COP and delta-COP), an ARF-GAP (Glo3p), a multi-spanning integral membrane protein that binds to coatomer (Erv29p) and a novel evolutionarily conserved protein called Vps74p, which I have shown here to interact with coatomer as well as with SNARE proteins. While these proteins all likely contribute to quality control and negative regulation of transport vesicle formation in the Golgi they do not all act via the same pathway. In summary, my study has resulted in the identification of a novel factor involved in membrane traffic (Vps74p), identified components of the protein machines that negatively regulate transport vesicle biogenesis in the Golgi and shed light on the underlying dynamics and adaptability of membrane trafficking pathways in higher eukaryotes.