Vps45 Regulates Tlg2 and Snc Levels Reference [12] [5] This study This study Table 1. Yeast strains used in this study. Strain SF838-9D NOzY1 RMY8 MSY002 Genotype MATa ura3–52 leu2–3, 112 his4–519 ade6 gal2 pep4-3 MATa ura3–52 leu2–3, 112 his4–519 ade6 gal2 pep4-3 vps45D::Kanr MATa ura3–52 leu2–3, 112 his3–D200 trp1–D901 suc2–D9 lys2–801 pep4–DH3 MATa ura3–52 leu2–3, 112 his3–D200 trp1–D901 suc2–D9 lys2–801 pep4–DH3 vps45D::Kanr RMY8 and MSY002 are congenic to SEY6210 [11]. NOzY1 is congenic to SF838-9D. doi:10.1371/journal.pone.0049628.t001 function in SNARE complex assembly, but are a secondary consequence of the reduced levels of Snc2 in these cells. Work is currently underway to identify mutant versions of Vps45 that stabilise Tlg2, but not Snc2, and also versions of Snc2 that are not subject to downregulation in the absence of Vps45. Such tools will allow us to address many important questions that arise from the present study, such as what factors determine whether a particular Snc participates in an exocytic or an endosomal SNARE complex. Here, we have demonstrated that Vps45 plays a role in regulating the cellular levels of, not only its syntaxin (Tlg2) but also its v-SNARE (Snc) binding partners. Furthermore, we have shown that this reduction in cellular Snc levels correlates with the impaired growth phenotypes seen for vps45 mutant cells. Prior to this study, this had been a puzzling observation since these growth phenotypes are not displayed by cells lacking Tlg2. Growth phenotypes are associated with loss of Vps45 orthologues in other organisms: in addition to endocytic defects, vps-45 mutation in Caenorhabditis elegans results in temperature sensitive lethality [37], and RNAi knockdown of AtVPS45 in Arabidopsis thaliana results in stunted growth [23]. Further work is required to investigate whether these are due to reduced levels of exocytic v-SNAREs. In addition, this study describes how cells may regulate levels of SNARE proteins as a mechanism to control membrane trafficking pathways. Materials and Methods Yeast Strains and Plasmids Yeast strains and plasmids used in this study are listed in Tables 1 and 2. The Class D yeast mutants used, in addition to NOzY1 (Table 1; vps45D), in Figure 3 are all congenic to SF838- 9D (Table 1) and are as follows: pep12, SGY39 [38]; vps15, mut.2066 [15]; vps21, SGY79 [39]; vps34, mut.223 [15]. Yeast were grown either in rich media [1% (w/v) yeast extract, 1% (w/v) Table 2. Plasmids used in this study. peptone, 2% (w/v) glucose: YEPD] or standard minimal medium with appropriate supplements [40]. RMY8 was constructed from SEY6210 [11] using pLO2010 as previously described [41]. MSY002 was then constructed from RMY8 using pNOzY13 to disrupt VPS45 as described [5]. pCOG054 encoding an HA- tagged version of Sncp was constructed using PCR to amplify genomic sequence of SNC2 with ,500 bp up stream and ,300 bp down stream with the following oligonucleotides (156; 59- TTAATACGAACAAAATAAAAATATG-39 AND 157; 59-AA- GACGGCCACTAAAACTGATG-39). The resultant product was cloned into pCR2.1-TOPO (Invitrogen), sequenced and sub- sequently subcloned (using the KpnI and XhoI sites contained within the oligonucleotides) into pRS426 [42]. Site directed mutagenesis was then used to introduced sequence encoding an HA-epitope tag immediately after the start codon of SNC2. Antibodies Polyclonal antibodies that specifically recognise the Snc v- SNAREs, Tlg2, Vps45 and Pgk1 and have been described previously [5,14,28]. Antibodies against the HA (3F10) epitope tags were from Sigma. Immunoblot Analysis of Yeast Cell Lysates Yeast cells were grown in standard minimal media (SD, lacking amino acids where appropriate for plasmid selection) [40]. 10 OD600 equivalents were harvested from cultures in early log phase and vortexed in 200 ml of Laemmli sample buffer in the presence of glass beads (425–600 mm, acid washed). Glass beads were allowed to settle during a 10 min incubation at 65uC and 10 ml of the lysate was loaded per lane of a 10% SDS-polyacrylamide gel. Proteins were next transferred to nitrocellulose for immunoblot analysis with antibodies as indicated. Immunoreactive proteins were visualized by detection with an anti-rabbit/mouse IgG horse radish peroxidase linked Plasmid pCOG054 pCOG070 pCOG071 pCOG072 YEplac195 pNB706 pNB707 pNB708 YCplac111 doi:10.1371/journal.pone.0049628.t002 Description Yeast expression plasmid (2 m, URA3) encoding HA-Snc2 Yeast expression plasmid (2 m, URA3) encoding wild-type HA-Vps45 As pCOG070, encoding HA-Vps45L117R As pCOG070, encoding HA-Vps45W244R Parent vector of pCOG070/1/2 Yeast expression plasmid (CEN, LEU2) encoding wild-type HA-Vps45 As pNB706, encoding HA-Vps45L117R As pNB706, encoding HA-Vps45W244R Parent vector of pNB706, pNB707 and pNB708 Reference This study [30] [7] [7] [7] [7] [7] [7] [30] PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49628