<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

<i>rsp5</i> and <i>bre5</i> mutations have additive effect on growth and mislocalization of GFP-Rer1.

<p>(<b>A</b>) <i>bre5Δ</i> and <i>HA-rsp5-1</i> mutations show weak genetic interaction. Strains <i>doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1</i> and <i>doa4Δ bre5Δ HA-rsp5-1</i> were transformed with plasmid encoding <i>DOA4</i>. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (<b>B</b>) <i>doa4Δ bre5Δ HA-rsp5-1</i> mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.</p

Retrograde trafficking from the Golgi to the ER is impaired in double <i>arp2-1 rsp5-1</i> and <i>arp2-1 ret1-1</i> mutants.

<p>(<b>A-C</b>) Analysis of transport from the Golgi to ER in spore clones from cross <i>arp2-1</i> × <i>ret1-1.</i> (<b>A</b>) Defect of GFP-Rer1 trafficking in <i>arp2-1 rsp5-1</i> mutants. Spore clones were transformed with plasmid encoding <i>GFP-RER1</i>. Transformants were grown and analyzed similarly as spore clones in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g004" target="_blank">Figure 4B</a>. (<b>B</b>) Double mutant <i>arp2-1 rsp5-1</i> is not sensitive to neomycin. Spore clones were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (<b>C</b>) Secretion of Kar2 is enhanced in <i>arp2-1 rsp5-1</i> mutant. Spore clones were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting Kar2 were identified by Western blotting with anti-Kar2 antibody. (<b>D–F</b>) Analysis of transport from the Golgi to ER in spore clones from cross <i>arp2-1</i> × <i>ret1-1.</i> (<b>D</b>) Defect of GFP-Rer1 trafficking in <i>arp2-1</i> × <i>ret1-1</i> mutant. The spore clones were transformed with plasmid encoding <i>GFP-RER1</i>. Transformants were grown and analyzed similarly as spore clones in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g004" target="_blank">Figure 4B</a>. (<b>E</b>) Double mutant <i>arp2-1 ret1-1</i> is sensitive to neomycin. The sensitivity to neomycin was tested as in B. (<b>F</b>) Secretion of Kar2p is enhanced in <i>arp2-1 ret1-1</i> mutant. The secretion was assayed as in C.</p

CPY secretion and Sna3-GFP trafficking and invertase activity are the same in <i>ret1-1 rsp5-1</i> mutant and in single <i>rsp5-1</i>.

<p>(<b>A</b>) Double mutants <i>ret1-1 rsp5-1</i> and <i>sec27-1 rsp5-1</i> do not secrete more CPY compared with the single <i>ret1-1</i>, <i>sec27-1</i> or <i>rsp5-1</i> mutants. Spore clones (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a>) were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting CPY were identified by Western blotting with anti-CPY antibody. (<b>B</b>) Sna3-GFP trafficking defect caused by <i>rsp5-1</i> mutation is not augmented by <i>ret1-1</i>. Plasmid encoding Sna3-GFP was transformed into spore clones as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2A</a>. Transformants were grown to mid logarithmic phase on SC -ura at 30°C or shifted to 35°C for 1 hour. Sna3-GFP was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole and viewed with Nomarski optics (NOM). (<b>C</b>) Whole cell lysates form transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g003" target="_blank">Figure 3B</a> were analyzed by Western blotting with anti-GFP antibody. Percentage of Sna3-GFP in total GFP signal (GFP and Sna3-GFP) is given. (<b>D</b>) Invertase activity was assayed in spore clones as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2A</a>. Cultures were grown to mid logarithmic phase at 30°C and shifted or not to 35°C for 30 minutes. The proportion between activity of secreted invertase to the total invertase activity (invertase secretion index) is shown.</p

Overexpression of ubiquitin suppresses <i>ret1-1 rsp5-1</i> mutant defects.

<p>(<b>A</b>) Growth defect of <i>ret1-1 rsp5-1</i> mutant is suppressed by overexpression of ubiquitin or its variants. <i>ret1-1 rsp5-1</i> mutant was transformed with empty vector [-] or with plasmids encoding wild type ubiquitin [UBI], ubiquitin with only single lysine 48 [K48] or 63 [K63] present and all other lysines replaced with arginine. Serial 1∶10 dilutions of transformants were spotted on YPD medium and incubated for 2 days at indicated temperatures. (<b>B</b>) Localization of GFP-Rer1 to vacuole in <i>ret1-1 rsp5-1</i> mutant is suppressed by overexpression of ubiquitin. Transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6A</a> were additionally transformed with plasmid encoding GFP-Rer1 and were grown on SC -ura -leu at 28°C. Expression of ubiquitin variants was induced by addition of 100 µM CuSO₄ for 2 hours before observations. GFP-Rer1 was localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>C</b>) Whole cell lysates form transformants from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6B</a> were analyzed by Western blotting with anti-GFP antibody. Percentage of GFP-Rer1 in total GFP signal (GFP and GFP-Rer1) in each lane is given. (<b>D</b>) Wild type or <i>rsp5-1</i> mutant were transformed with a plasmid expressing <i>His₆-UBI</i> and with empty vector [-] or a plasmid encoding GFP-Rer1. Transfromants were grown to mid-logarithmic phase at 30°C. Expression of ubiquitin was induced by addition of 100 µM CuSO₄ for 2 hours before cultures were shifted or not to 37°C for 1 hour. His₆-Ubi was pulled down on Ni-NTA beads. Total fraction and fraction bound to beads were analysed by Western blotting with anti-GFP antibody. (<b>E</b>) Fusion protein Ub-GFP-Rer1 is targeted to vacuole. The <i>rer1-1 rsp5-1</i> strain was transformed with plasmid encoding one of the fusions <i>GFP-RER1</i> or <i>Ub-GFP-RER1</i>. The GFP-Rer1 and Ub-GFP-Rer1 proteins were localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>F</b>) Ub-GFP-Rer1 protein is expressed. Total protein extracts from the same transformants as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g006" target="_blank">Figure 6E</a> were analyzed by Western blotting with anti-GFP antibody.</p

Retrograde trafficking from Golgi to ER is impaired in double <i>ret1-1 sla1Δ</i> mutant, but not in <i>rsp5-19 sla1Δ</i>.

<p>(<b>A</b>) Negative genetic interaction between <i>ret1-1</i> and <i>sla1Δ</i> and no such a genetic interaction between <i>sec27-1</i> and <i>sla1Δ</i> mutations. Serial 1∶10 dilutions of spore clones from crosses <i>ret1-1</i> × <i>sla1Δ</i> (JK82-4B × <i>sla1Δ</i>) and <i>sec27-1</i> × <i>sla1Δ</i> (JK84-3C × <i>sla1Δ</i>) were spotted on YPD medium or YPD containing 1 mM neomycin (NEO) and incubated for 2 or 3 days at indicated temperatures. (<b>B</b>) The spore clones from crosses <i>ret1-1</i> × <i>sla1Δ</i> were transformed with plasmid expressing GFP-Rer1 and localization of GFP was monitored by fluorescence. (<b>C</b>) Sla1 is in complex with COPI proteins. The <i>sla1Δ</i> mutant was transformed with empty vector ([-]) or with plasmid expressing <i>HA-SLA1</i>. Protein extracts were prepared from transformants and HA-Sla1 was immunoprecipitated with anti-HA antibody. The total protein extracts (T) and immunprecipitated materials (IP) were analyzed by Western blotting with anti-HA and anti-COPI antibody. (<b>D</b>) <i>rsp5-19</i> and <i>sla1Δ</i> mutations have no additive effect on GFP-Rer1 localization. The spore clones from crosses <i>rsp5-19</i> × <i>sla1Δ</i> were transformed with plasmid expressing GFP-Rer1 and localization of GFP was monitored by fluorescence.</p

Retrograde trafficking from Golgi to ER is impaired in double <i>ret1-1 rsp5-1</i> mutant.

<p>(<b>A</b>) Double mutant <i>ret1-1 rsp5-1</i> is sensitive to neomycin. Spore clones from <i>ret1-1</i> × <i>rsp5-1</i> cross (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a>) were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (<b>B</b> and <b>C</b>) Defect of GFP-Rer1 trafficking in <i>ret1-1 rsp5-1</i> and <i>sec27-1 rsp5-1</i> mutants. Spore clones (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039582#pone-0039582-g003" target="_blank">3B</a>) from <i>ret1-1</i> × <i>rsp5-1</i> and <i>sec27-1</i> × <i>rsp5-1</i> crosses were transformed with plasmid encoding <i>GFP-RER1</i>. Transformants were grown on SC -ura at 28°C. GFP-Rer1 was localized by fluorescence (GFP) and cells were viewed with Nomarski optics (NOM). (<b>D</b>) <i>rsp5-1</i> mutation is responsible for additional defect in GFP-Rer1 trafficking caused by <i>ret1-1</i>. Centromeric vector encoding <i>HA-RSP5</i> or empty vector ([-]) were transformed into <i>ret1-1 rsp5-1</i> expressing <i>GFP-RER1</i>. Whole cell protein extracts from transformants were analysed by Western blotting with anti-GFP antibody. (<b>E</b>) Secretion of Kar2p is enhanced in <i>ret1-1 rsp5-1</i> mutant. Spore clones from <i>ret1-1</i> × <i>rsp5-1</i> cross (as in B) were grown at 28°C in YPD, transferred to fresh medium and incubated at 28°C for 1 h. Whole cell protein extracts and proteins TCA-precipitated from medium were analyzed by Western blotting with anti-Kar2 and anti-PGK antibody. The latter was to control cell integrity. (<b>F</b>) HA-Rsp5 binds COPI complex. The extracts from <i>rsp5Δ</i> strain transformed with empty vector ([-]) or with centromeric plasmid YCpHA-RSP5 ([<i>HA-RSP5</i>]) were used for immunoprecipitation using anti-HA antibody (16B12). Total extracts (T) and immunoprecipitated fraction (IP) were analysed by Western blotting with anti-HA and with anti-coatomer antibody.</p

Two dimensional chromatography of glycerophospholipids.

<p>The levels of all major glycerophospholipids were diminished by treatment with simvastatin. Panels: 1, 3, 5 glycerophospholipids from cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Panels 2, 4, 6 glycerophospholipids from simvastatin treated cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Abbreviations: PC phosphtidylcholine, PE phosphtidylethanolamine, PS phosphatidylserine, PI phosphtidylinositol, PA phosphtidic acid, LP lysoglycerophospholipid, FA fatty acid, NL neutral lipids.</p

Nile Red staining of yeast lipid particles.

<p>Visualization of membranes composed of glycerophospholipids (red emission) and neutral lipids, triacyglycerols and steryl esters, in lipid particles (green emission). Cells were cultured overnight. The media were then supplemented with either 100 µM simvastatin or buffer and the cells were further grown with shaking for two hours at 30°C. To localize neutral lipids and glycerophospholipids in yeast cells, Nile Red staining was performed. Horizontal panels: upper glycrophospholipids at 543 nm excitation and 610 nm emission, middle neutral lipids at 488 nm excitation and 515/530 nm emission, lower merge of above panels. Vertical panels: B cells cultivated in buffer, S cells incubated for 2 h in buffer with simvastatin.</p

Decrease in sterols and squalene after simvastatin treatment.

<p>Lipids extracted from yeast cells were subjected to alkaline hydrolysis, purified and analysed by GC/MS.</p><p>Wt, wild-type yeast; H, yeast harbouring wild-type <i>hHMGR</i> gene; h, yeast harbouring the mutated <i>hHMGR</i> gene.</p