Regulation of membrane fusion by Tlg2p & Vps45p through the endosomal system of Saccharomyces cerevisiae

QH301 Biology

MacDonald, Chris ; Bryant, Nia J.

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: SNARE, Sec1/Munc18p, membrane fusion;   

Others  :  http://theses.gla.ac.uk/1403/1/2009macdonaldphd.pdf

来源: University of Glasgow

【 摘 要 】

SNARE proteins are essential components of the machinery that facilitates membrane fusion in eukaryotic cells.SNARE proteins are subject to multiple levels of regulation, one of which is imbedded in the syntaxin (Qa-SNARE) molecule.It has been demonstrated that some syntaxins adopt a closed conformation, whereby an autonomously folded N terminal domain (the Habc domain) forms intramolecular contacts with the SNARE domain; this conformation precludes complex assembly.The Sec1p / Munc18 (SM) family are a conserved group of proteins that regulate membrane fusion through interactions with their cognate syntaxins.Formulation of unifying hypotheses describing how SM proteins function has been problematic, primarily due to the multiple modes of interaction that have been characterised for different members of this family binding to their cognate SNARE proteins. The yeast SM protein Vps45p regulates membrane fusion through the trans-Golgi / late endosomal system, and interacts directly with the syntaxin (Tlg2p) and the v-SNARE (Snc2p) proteins.Vps45p also binds to the assembled SNARE complex of Tlg2p, Vti1p, Tlg1p and Snc2p.In this thesis I demonstrate that the Habc domain of Tlg2p has an inhibitory effect on SNARE complex formation.This is an important finding, as whether or not Tlg2p adopts a closed conformation has hitherto been controversial.Furthermore, I have demonstrated that the inhibitory effect of the Habc domain on complex formation can be alleviated by Vps45p in vitro.In addition to investigating the functional significance of Vps45p’s interaction(s) with Tlg2p, I have also investigated binding of the SM protein to the v-SNARE Snc2p.I have demonstrated that the affinity of Vps45p for Snc2p is weaker than either of the modes of interaction characterised between Vps45p and Tlg2p.Finally, I have developed an in vitro fusion assay to enable us to dissect the functional significance of the various interactions that Vps45p displays with its cognate SNARE proteins.