the vacuolar hydrolase carboxy peptidase Y (CPY) [15]. This suggested that there are multiple trafficking steps in the Vps pathway from the TGN to the vacuole, and that gene products affected in mutants of each of the classes act in the same process or membrane trafficking step. Further studies have revealed that this in indeed the case, and in many cases proteins encoded by genes in the same class function as part of a multi-protein complex. This is perhaps best exemplified by the class E vps mutants [15]. Class E vps mutants accumulate an exaggerated form of the endosomal compartment through which biosynthetic and endocytosed material transits en route to the vacuole [15]. Biochemical, morphological and genetic studies have demonstrated that the gene products represented by Class E vps mutants all function in sorting ubiquitinated proteins into multivesicular bodies as part of the ESCRT (endosomal sorting complex required for transport machinery [16,17]. Similarly, the 4 class C proteins (Vps11, Vps16, Vps18 and Vps33) form the core of the CORVET and HOPS tethering complexes that function at endosomal and vacuolar membranes respectively [18]. VPS45 is a class D gene [15]. Class D Vps proteins, including Vps45 and the regulatory and catalytic subunits of PI-3 kinase (Vps15 and Vps34), are required for traffic between the TGN and endosomes [19]. Interaction between Vps45 and Tlg2 is not only important for regulating the function of Tlg2, but also stabilises the syntaxin, with cellular levels of Tlg2 being dramatically reduced in cells lacking Vps45 [5,20]. This feature appears to be conserved across species, and also across different membrane trafficking steps, as several SM proteins have been shown to stabilise their syntaxins [21,22,23,24]. Controlling cellular levels of SNARE proteins represents another mechanism by which membrane fusion may be regulated. Like many SM proteins, Vps45 also binds directly to its cognate v-SNARE [7], and we therefore set out to investigate whether v-SNARE levels are also regulated by binding to the SM protein. We now report that, like those of Tlg2, levels of the Snc v-SNARE are regulated by Vps45. As well as assembling into Tlg2p-containing SNARE complexes to regulate endosomal traffic, the Snc2 v-SNAREs also participate in exocytic Sso-containing SNARE complexes required for fusion of secretory vesicles with the plasma membrane; a trafficking pathway required for growth [25,26]. These data explain why vps45D cells are unique amongst the Class D vps mutants in that they display multiple growth phenotypes and reveal the impor- tance of regulating cellular levels of SNARE proteins. Results and Discussion Vps45p Regulates Levels of its Cognate SNARE Binding Partners Cells lacking the SM protein Vps45 have reduced levels of its cognate syntaxin Tlg2 [5,7,20,27]. In addition to binding Tlg2, Vps45 also binds the v-SNARE Snc2 [7]. To address whether absence of Vps45 affects cellular levels of Snc2, we used immunoblot analysis to assess the levels of an HA-tagged version of Snc2 in wild-type and vps45D mutant cells. Figure 1A shows that cells lacking Vps45 (vps45D) contain substantially less HA-Snc2 than wild-type cells harbouring the same HA-Snc2 expression construct (pCOG054). Analysis of two different strain backgrounds demonstrates that cellular levels of HA-Snc2 produced from the 2m-plasmid in vps45D mutant cells are approximately 33% of those found in their congenic wild-type strains (Fig. 1A). To extend these studies and look at levels of the endogenous v-SNARE, we used polyclonal antiserum raised against residues 72–86 (GFKRGANRVRKQMWW) of Snc2 [28]. This region of the protein shares 87% identity with the analogous region of Snc1 (residues 73–87; GFKRGANRVRKAMWY), whose functions are Vps45 Regulates Tlg2 and Snc Levels Figure 1. Deletion of VPS45 results in reduced cellular levels of Tlg2 and the Snc2 proteins. Proteins contained within cell lysates prepared from wild-type (SF838-9D and RMY8; lanes 1 and 3) and congenic vps45D mutant cells (NOzY1 and MSY002; lanes 2 and 4) were separated using SDS-PAGE before being transferred to nitrocellulose. The resulting filters were probed using antibodies that recognise Vps45, Tlg2, the HA-epitope [for HA-tagged Snc2 expressed from pCOG054] in (A), the Snc v-SNAREs in (B), and Pgk1 (phosphoglycerare kinase, which was included as a loading control). N.B. The cells used for (A) harbour pCOG054 whereas those used in (B) do not. doi:10.1371/journal.pone.0049628.g001 redundant with Snc2 for cellular processes including exo- and endocytosis [26,29]. Immunoblot analysis using this antiserum demonstrates that cellular levels of endogenous Snc v-SNAREs in cells lacking Vps45 (vps45D) are approximately 65% of those in congenic wild-type cells (Fig. 1B). The data presented in Figure 1 indicate that cellular levels of the v- SNARE Snc proteins and the syntaxin Tlg2 are regulated by their cognate SM protein Vps45p. To test this hypothesis we set out to increase the cellular levels of Vps45 and ask whether this leads to PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49628