Figure 2. Elevated cellular levels of Vps45 result in a concom- itant increase in Tlg2 and Snc2 levels. (A) Proteins contained within cell lysates were prepared from wild-type cells (SF838-9D) harbouring either the empty vector YEplac195 (lane 1) or the multicopy 2 m plasmid pCOG070 encoding HA-Vps45 (lane 2), were separated using SDS-PAGE before being transferred to nitrocellulose. The resulting filters were probed using antibodies that specifically recognise the HA- epitope, Vps45, Tlg2, Snc2 and Pgk1 as indicated. (B) Proteins contained within cell lysates were prepared as described in (A) from vps45D mutant cells (NOzY1) producing either no Vps45 (carrying the empty vector YEplac195; lane 1), wild-type HA-Vps45 (lane 2) or HA-Vps45L117R (lane 3) from multicopy 2 m plasmids (pCOG070 and pCOG071 respectively). doi:10.1371/journal.pone.0049628.g002 a concomitant increase in Tlg2 and Snc levels. This was achieved by expressing VPS45 from a multicopy plasmid in wild-type cells (Fig. 2A). Cells harbouring pCOG070, encoding HA-tagged Vps45 [7], contain approximately 2.5-fold more Vps45 than those carrying Vps45 Regulates Tlg2 and Snc Levels the parental vector YEplac195 [30], and also contain increased levels of Tlg2 and the Snc v-SNAREs (both $2-fold). Selective Stabilisation of Snc by a Mutant Version of Vps45 Abrogated for Tlg2-Binding We have previously reported that a version of Vps45 (Vps45L117R) harbouring a mutation that abrogates binding of the SM protein to the N-terminal peptide of Tlg2 [7,8] does not stabilise Tlg2, demonstrating that this mode of binding between Vps45 and Tlg2 stabilises the syntaxin [27]. The L117R mutation disrupts the hydrophobic pocket-binding site situated on the outer surface of domain-1 of the arch-shaped SM protein [7,8]. The binding site for Snc2 is distinct from this hydrophobic pocket, demonstrated by the observation that the Vps45L117R mutant binds the cytosolic domain of Snc2 in vitro in a manner indistinguishable from wild-type Vps45 [7]. Figure 2B shows that, as previously reported [27], vps45D cells expressing Vps45L117R have reduced levels of Tlg2 compared to the same cells expressing wild-type Vps45. In contrast, Snc levels are comparable in vps45D cells expressing either Vps45L117R or wild-type Vps45 (both substantially higher than in the same cells carrying empty vector; Fig. 2B). These data indicate that binding to Vps45 is important for the regulation of cellular levels of its cognate SNARE binding partners. This is an important observation as it provides the cell with a mechanism to ensure that the SNAREs are not present in excess compared to their regulatory SM protein. Were such a situation to arise it could prove catastrophic for the cell allowing SNARE complexes to form in an unregulated manner. Growth Phenotypes of vps45 Mutant Cells Correlate with Reduced Cellular Levels of Snc v-SNAREs VPS45 is a class D VPS gene [15]. Class D VPS gene products, 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]. The original VPS genes, identified through genetic screens for mutants that missort CPY [11,12] are not essential due to the existence of multiple pathways for delivery of macro- molecules to the vacuole [31,32]. Consistent with this, most vps mutants do not show growth defects under normal laboratory conditions. One exception to this are vps45 mutants [13,14]. Figure 3A shows that cells lacking Vps45p have significantly longer doubling time, and slower growth rates, than congenic wild-type cells, whereas other class D vps mutants do not. This difference in growth phenotype between vps45 mutant cells and other class D vps mutants is also illustrated by the observation that vps45 mutants display a marked growth defect at 39uC whereas other class D mutants tested do not (Fig. 3B). These observations are surprising, as removal of different components involved in the same transport step would be expected to result in similar phenotypes. Although not identified in the original vps genetic screens, tlg2 mutants are vps mutants in that they missort the vacuolar hydrolase CPY [33]. Like other VPS genes, TLG2 is not essential. Similarly, Vam3, the syntaxin that regulates traffic at the vacuolar membrane is also not essential [34,35]. In contrast SNAREs required for membrane traffic to the cell surface are essential [36]. As well as participating in endosomal Tlg2-containing SNARE complexes [33], Snc2 is also required for the fusion of secretory vesicles with the plasma membrane for exocytosis [26]. Exocytosis is required for growth, and as such snc mutants are inviable [26]. We hypothesized that the growth phenotype of vps45D cells might be due to their reduced levels of Snc v-SNAREs and took advantage of our finding that expression of the Vps45L117R mutant in vps45D cells selectively restores the levels of Snc2 (Fig. 2) to test this. Figures 4A and 4B show that expression of Vps45L117R PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49628