Vps45 Regulates Tlg2 and Snc Levels Figure 3. vps45 mutants display growth defects not found in other class D vps mutants. (A) The specific growth rate (m) and doubling time of wild-type cells (SF838-9D) and congenic class D vps mutants (pep12, vps15, vps21, vps34, vps45) was determined during logarithmic growth in YEPD. Data represent the mean 6 SEM of 6 independent sets of cells. *, P,0.05 versus wild-type. (B) Cells were grown for 16 h at 30uC in a shaking incubator, before being diluted to an OD600 of 0.2 and grown for 2 further doublings. 10 OD600 equivalents of cells were harvested and resuspended in 1 ml dH2O. 5 ml of a 1/10 and a 1/100 dilution of this suspension was spotted onto solid media and incubated at either 30uC or 39uC for 3 days. doi:10.1371/journal.pone.0049628.g003 complements the reduced growth rate, longer doubling time and temperature-sensitive growth phenotypes of vps45D cells as efficiently as wild-type Vps45. We have also previously characterised a second mutant of Vps45p, Vps45W244R, which gives a dominant negative phenotype for sorting of vacuolar hydrolases [7,27]. The CPY missorting phenotype of this dominant negative mutant is dose-dependent and can be abrogated by co-expression of either wild-type Vps45 or Snc2, but not Tlg2 [7]. This suggests that Vps45W244R disrupts endosomal trafficking by titrating out the v-SNARE Snc2. Expression of Vps45W244R in vps45D cells does not complement the CPY missorting phenotype observed in vps45D cells [7,27]. Figure 4B shows that expression of the mutant Vps45W244R in vps45D cells partially complements the temperature-sensitive growth phenotype of vps45D cells, restoring growth at 39uC. Moreover, Figure 4C demonstrates that co- expression of HA-Snc2 from a multicopy plasmid with Vps45W244R fully restores growth to levels seen with Vps45p and Vps45pL117R mutants. These data indicate that the growth defects of vps45D cells are independent of the CPY missorting phenotype as they can be overcome by increasing Snc2 levels in cells containing a non- functional version of Vps45p. It is important to remember that SM protein function in SNARE- mediated membrane traffic extends beyond a role in maintaining cellular levels of its binding partners. This is demonstrated by our previous work showing that restoration of Tlg2 levels in vps45D cells is not sufficient to complement the vacuolar trafficking defects of these cells, and has also been reported for other SM/Syntaxin pairs including syntaxin-1/Munc18-1 [5]. The multiple functions of SM proteins complicates the challenge of understanding the role(s) of this protein family in vivo [2–4]. The observation that increasing levels of Snc2 complements growth phenotypes of vps45D cells likely reflects the role of the exocytic SM protein, Sec1p, in regulating Snc- containing SNARE complexes at the plasma membrane, as oppose to Vps45 in the endosomal system v-SNAREs [4]. Further studies will be required to ascertain whether this is the case as alternative/ compensatory mechanisms or pathways may complicate genetic analyses in vivo. The data presented here indicate that the growth phenotypes of cells lacking Vps45 are not related to the SM proteins PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49628