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

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

Yeast strains used in this study.

<p>RMY8 and MSY002 are congenic to SEY6210 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049628#pone.0049628-Robinson1" target="_blank">[11]</a>. NOzY1 is congenic to SF838-9D.</p

Elevated cellular levels of Vps45 result in a concomitant increase in Tlg2 and Snc2 levels.

<p>(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 <i>vps45</i>Δ mutant cells (NOzY1) producing either no Vps45 (carrying the empty vector YEplac195; lane 1), wild-type HA-Vps45 (lane 2) or HA-Vps45_L117R (lane 3) from multicopy 2 μ plasmids (pCOG070 and pCOG071 respectively).</p

Deletion of VPS45 results in reduced cellular levels of Tlg2 and the Snc2 proteins.

<p>Proteins contained within cell lysates prepared from wild-type (SF838-9D and RMY8; lanes 1 and 3) and congenic <i>vps45</i>Δ 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.</p

Vps45_L117R complements growth phenotypes of vps45Δ cells.

<p>(A) The specific growth rate (μ) and doubling time of wild-type (SF838-9D) and congenic <i>vps45</i>Δ mutant (NOzY1) cells producing either no HA-Vps45 (carrying the vector YEplac195), wild-type HA-Vps45 (<i>VPS45</i>) or HA-Vps45_L117R (L117R) from pCOG070 and pCOG071 respectively (WT and <i>vps45</i>Δ 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 <i>vps45</i>Δ. (B) Wild-type cells (SF838-9D) harbouring YEPlac195 or pCOG070 (HA-Vps45), and congenic <i>vps45</i>Δ mutant cells (NOzY1) harbouring plasmids YEplac195 (empty vector), pCOG070 (HA-Vps45), pCOG71 (HA-Vps45_L117R) or pCOG072 (HA-Vps45_W244R) were analysed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049628#pone-0049628-g003" target="_blank">Figure 3B</a>. (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 <i>vps45</i>Δ mutant cells (NOzY1) harbouring YCplac111 (empty vector) or pNB706 (HA-Vps45), pNB707 (HA-Vps45_L117R) or pNB708 (HA-Vps45_W244R) were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049628#pone-0049628-g003" target="_blank">Figure 3B</a>.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Effects of an exercise intervention for older heart failure patients on caregiver burden and emotional distress

BACKGROUND: The impact of exercise programmes for heart failure on those close to the patient is largely unknown. We examined the effect of a hospital and home-based exercise intervention on burden, anxiety and depression of informal caregivers. DESIGN: The study was a randomized, controlled trial. Heart failure patients were randomized to a seated 12-week hospital-based exercise programme. Caregiver measures were gathered at baseline, 3 months later and 6 months following baseline. METHODS: Sixty caregivers (mean age 63.4 years, 65% female) of heart failure patients (n = 82, mean age 80.5 years, 44% female) participating in a trial of an exercise intervention were recruited. Caregiver burden, anxiety and depression were assessed. RESULTS: There were no differences in caregiver burden, depression or anxiety between the two groups of caregivers at baseline (caregiver burden, patient control 33.1 versus patient exercise 34.1; anxiety 4.1 versus 5.5; depression 2.8 versus 3.8). At 3 months there were no differences between caregivers in the two groups on outcomes. At 6-month follow-up caregivers of heart failure patients in the exercise group had burden scores that were significantly worse than the control group. There were no differences between the carers of exercise and control groups in anxiety and depression. Levels of anxiety and depression in the entire carer sample were marginally higher than reference values in a healthy non-clinical sample. CONCLUSION: The present exercise interventions for frail older patients did not benefit caregivers and was associated with an increase in caregiver burden. We suggest that future exercise interventions for heart failure patients should actively incorporate informal caregivers into research designs

Functional homology of mammalian syntaxin 16 and yeast Tlg2p reveals a conserved regulatory mechanism

Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Δ mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed