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. 2012;7(11):e49628.
doi: 10.1371/journal.pone.0049628. Epub 2012 Nov 14.

The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Scott G Shanks  1 , Lindsay N Carpp, Marion S Struthers, Rebecca K McCann, Nia J Bryant
Affiliations

The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Scott G Shanks et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 vps45Δ 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.
Figure 2
Figure 2. Elevated cellular levels of Vps45 result in a concomitant 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 μ 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 vps45Δ 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 μ plasmids (pCOG070 and pCOG071 respectively).
Figure 3
Figure 3. vps45 mutants display growth defects not found in other class D vps mutants.
(A) The specific growth rate (μ) 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 ± SEM of 6 independent sets of cells. *, P<0.05 versus wild-type. (B) Cells were grown for 16 h at 30°C 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 µl of a 1/10 and a 1/100 dilution of this suspension was spotted onto solid media and incubated at either 30°C or 39°C for 3 days.
Figure 4
Figure 4. Vps45L117R complements growth phenotypes of vps45Δ cells.
(A) The specific growth rate (μ) and doubling time of wild-type (SF838-9D) and congenic vps45Δ mutant (NOzY1) cells producing either no HA-Vps45 (carrying the vector YEplac195), wild-type HA-Vps45 (VPS45) or HA-Vps45L117R (L117R) from pCOG070 and pCOG071 respectively (WT and vps45Δ cells producing no HA-Vps45 carried the vector YEplac195) was determined during logarithmic growth in minimal media. Data represent the mean ± SEM of 6 independent sets of cells. §, P<0.05 versus WT; #, P<0.05 versus vps45Δ. (B) Wild-type cells (SF838-9D) harbouring YEPlac195 or pCOG070 (HA-Vps45), and congenic vps45Δ mutant cells (NOzY1) harbouring plasmids YEplac195 (empty vector), pCOG070 (HA-Vps45), pCOG71 (HA-Vps45L117R) or pCOG072 (HA-Vps45W244R) were analysed as described for Figure 3B. (C) All yeast strains in this panel harbour plasmid pCOG054, producing HA-Snc2 in addition to the following plasmids. Wild-type cells (SF838-9D) harbouring YCplac111 (empty vector) and congenic vps45Δ mutant cells (NOzY1) harbouring YCplac111 (empty vector) or pNB706 (HA-Vps45), pNB707 (HA-Vps45L117R) or pNB708 (HA-Vps45W244R) were analyzed as in Figure 3B.
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