The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae Scott G. Shanks, Lindsay N. Carpp, Marion S. Struthers, Rebecca K. McCann, Nia J. Bryant* Henry Wellcome Laboratory of Cell Biology, Institute of Molecular, Cell & Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom Abstract Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic. Citation: Shanks SG, Carpp LN, Struthers MS, McCann RK, Bryant NJ (2012) The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae. PLoS ONE 7(11): e49628. doi:10.1371/journal.pone.0049628 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received July 12, 2012; Accepted October 11, 2012; Published November 14, 2012 Copyright: 2012 Shanks et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the BBRSC (to NJB) and The Wellcome Trust (studentship to LNC). NJB is a Prize Fellow of the Lister Institute of Preventive Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nia.bryant@glasgow.ac.uk Introduction Spatial and temporal regulation of membrane traffic is essential for maintenance of one of the defining features of eukaryotic cells; intracellular compartmentalisation into discrete membrane bound organelles [1]. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are central to membrane traffic in all eukaryotes, with formation of SNARE complexes between SNAREs on opposing membranes being defined as the minimal requirement for membrane fusion [2]. SNARE complexes consist of 4 helical SNARE motifs contributed by members of the Qa (syntaxin), Qb, Qc (also called t-SNAREs) and R- (also called v-SNAREs) subfamilies [3]. Regulating SNARE complex assembly allows the cell to regulate membrane traffic. One family of proteins that regulate this process are the Sec1/Munc18 (SM) proteins [4]. Although their mechanism of action is not completely understood, it is clear that SM proteins are universally required for functional SNARE complex assembly [4]. We have previously characterised a role for the SM protein Vps45 in regulating SNARE-mediated membrane traffic through the endo- somal system of the yeast Saccharomyces cerevisiae [5,6,7]. Vps45 activates the syntaxin Tlg2 for entry into functional SNARE complexes with its partner t-SNAREs Tlg1, Vti1 and the v-SNARE Snc2 likely by facilitating a switch of Tlg2 from a closed (inactive) conformation to an open (active) state [5]. Vps45 binds directly to Tlg2, as well as to the v-SNARE, Snc2 and the assembled SNARE complex [6,7,8]. Of these interactions, Vps45’s binding to Tlg2 is the most thoroughly characterised. Like other SM proteins Vps45 has two distinct binding sites for its cognate syntaxin [8]. The higher- affinity binding site involves a conserved leucine residue in Vps45 that is predicted to form a hydrophobic pocket on the outer surface of the arch shaped SM protein [7]. This facilitates ‘mode 2’ binding to Tlg2 which requires the N-terminal 36 residues of the syntaxin, and is akin to the interaction captured by the Sly1/Sed5 crystal structure [7,9]. In addition, Vps45 binds Tlg2 through a second mechanism that does not involve the N-terminal peptide of the syntaxin, and while the structure of this interaction has not been solved, biochemical and biophysical studies indicate that it is akin to the interaction captured in the crystal structure of the neuronal Munc18a bound to Syntaxin1a; mode 1 binding [8,10]. The role that these two distinct binding modes between Vps45 and Tlg2 and indeed, between other SM proteins and their cognate syntaxins is not understood. Vps45 was originally identified through genetic screens for yeast defective in vacuolar protein sorting (vps mutants) [11,12], and has been shown to be required for delivery of proteins from the trans Golgi network (TGN) into the endosomal system [13,14]. Classification of the vps mutants into 6 groupings (A–F) based on vacuolar morphology suggested the existence of 6 separate processes whose individual perturbation result in missorting of PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49628