(A) Colocalization of 19S RP and 20S CP proteasome subunits in quiescent yeast cells

Cells coexpressing the indicated fusion proteins were imaged after 4 d of growth in YPDA at 30°C. GFP fluorescence is green and RFP fluorescence is red. Images are single focal planes. (B) Cells expressing Scl1p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. For each time point, cells displaying both the green and the red fluorescence were scored as indicated in the legend. For each time point, > 200 (two experiments; error bars show SD). Typical colocalization images corresponding to each type of proteasome localization pattern are shown on the right. Images are maximal projection of z stacks. Bars, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Reversible cytoplasmic localization of the proteasome in quiescent yeast cells"</p><p></p><p>The Journal of Cell Biology 2008;181(5):737-745.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396804.</p><p></p

(A) PSGs, as revealed by Pre6p-GFP fluorescence, were observed in cells grown for 4 d in different carbon sources containing rich media and in diploid cells grown in YPDA

For each condition > 200 (two experiments; error bars show SD). Typical images (maximal projection of z stacks) of cells displaying PSGs (Pre6p-GFP) are shown on the right. (B) Cells expressing Pre6p-GFP were grown in YPDA medium at 30°C, fixed, and stained with Alexa Fluor phalloidin to reveal F-actin–containing structures. Actively proliferating cells (left) displayed actin patches and cables (red) and a typical Pre6p-GFP nuclear localization. Quiescent cells displayed PSGs (green fluorescence) that did not colocalize with actin bodies (red). (C) Cells coexpressing Dcp2p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. Actively proliferating yeast cells displayed a typical Pup1p-RFP nuclear localization and small and discrete Dcp2p-GFP dots. In quiescent cells, Dcp2p-GFP localized in P-bodies that did not colocalize with PSGs. Images in B and C are single focal planes. Bars, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Reversible cytoplasmic localization of the proteasome in quiescent yeast cells"</p><p></p><p>The Journal of Cell Biology 2008;181(5):737-745.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396804.</p><p></p

Model of how a reduction in the hydrophobicity of subunit 9 permits its functional expression from nuclear DNA.

<p>When the hydrophobicity of subunit 9 is too high, the protein cannot cross the inner mitochondrial membrane (IM) and is degraded in the intermembrane space by the i-AAA protease. With reduced hydrophobicity, subunit 9 can cross the IM and is processed by the matrix processing peptidase (MPP), properly inserted into the IM, and assembled into ATP synthase (see text for details). OM, outer mitochondrial membrane; MTS, mitochondrial targeting sequence, TMH, transmembrane segment; TOM, translocase of the OM; TIM, translocase of the IM.</p

The <i>P. anserina Atp9</i> proteins are less hydrophobic than yeast Atp9p.

<p>A) Hydropathy profiles of the <i>PaAtp9-7</i> and <i>PaAtp9-5</i> proteins and yeast Atp9p, generated according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876-Kyte1" target="_blank">[54]</a> with a window size of 13. B) <i>P. anserina</i> strains expressing exclusively either <i>PaAtp9-7</i> (PaΔAtp9-5) or <i>PaAtp9-5</i> (PaΔAtp9-7) were constructed and ATP synthase was enriched from their mitochondrial extracts, separated by SDS-PAGE and silver-stained along with <i>WT</i> yeast ATP synthase. Positions of some ATP synthase subunits are indicated. The <i>PaAtp9</i>-5 protein is stained much more strongly than the <i>PaAtp9-7</i> protein, which may be due to the differences in their amino acid sequences.</p

Transcriptome profiles of yeast strains expressing <i>P. anserina Atp9</i> genes indicate functional OXPHOS and regulatory responses to the nuclear relocation of <i>ATP9</i>.

<p>For all genes in the yeast genome, the expression levels in AMY11 (expressing <i>PaAtp9-7</i>) are plotted against those of AMY10 <i>(PaAtp9-5)</i>, both displayed as log₂ ratios to <i>WT</i> expression levels; differentially expressed genes in the main functionally relevant categories are indicated by colours. The square formed by the grey lines delineates the boundaries of statistically significant expression differences (see <i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s007" target="_blank">Text S1</a></i>) between either strain and the <i>WT</i>; genes beyond the diagonal grey lines are differentially expressed between AMY10 and AMY11. For clarity, genes in the categories listed are only indicated if they were differentially expressed relative to <i>WT</i> in at least one strain. Categories were defined as follows: OXPHOS pathway - subunits (1/35 differentially expressed) and biogenesis factors (1/42); Retrograde pathway – transcriptional targets of the factors Gcn4p (29/126) and Rtg3p (6/31), plus <i>CIT2</i> and <i>CIT3</i>; Heat response - Gene Ontology (GO)-annotated “response to heat” genes (28/199); Morphology - Phd1p targets (23/81), plus GO “cell-cell adhesion” (2/4) and “cytokinesis, completion of separation” genes (6/11). All categories except OXPHOS were significantly enriched among differentially expressed genes (according to Fisher's exact test with multiple hypothesis testing correction, or to Model Gene Set Analysis (MGSA); see <i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s007" target="_blank">Text S1</a></i>).</p

Deletion of the yeast mitochondrial <i>ATP9</i> gene and resulting phenotypes.

<p>A) The mitochondrial <i>ATP9</i> gene was deleted and replaced with <i>ARG8^m</i> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s002" target="_blank">Figure S1</a> for details) in a wild-type strain lacking the nuclear <i>ARG8</i> gene. As a result, the <i>Δatp9</i> yeast grow on glucose (Glu) media lacking arginine (Arg) whereas the parental strain (<i>WT</i>) does not; in addition, <i>Δatp9</i> yeast cannot grow on glycerol (Gly). B) ATP synthase levels in <i>WT</i> and <i>Δatp9</i>. Isolated mitochondria were separated by BN-PAGE and western blotted with antibodies against Atp4p; <i>V</i>₁ and <i>V</i>_n respectively indicate monomeric and oligomeric forms of ATP synthase. C) Pulse labelling of proteins translated in mitochondria. Total proteins were prepared from cells incubated in the presence of ³⁵S methionine and cysteine as well as cycloheximide to inhibit cytosolic protein synthesis. Proteins (40,000 cpm per lane) were separated on 12% (Cox3p and Atp6p) or 17% (Atp9p and Atp8p) SDS-PAGE containing 6 M urea. D) Electron microscopy of <i>WT</i> (<i>a</i>) and <i>Δatp9</i> (<i>b–d</i>) cells grown in galactose (80 nm-thin sections); <i>m</i>, mitochondria; <i>Cr</i>, cristae; <i>Ib</i>, inclusion bodies; arrowheads in (<i>a</i>) point to <i>Cr</i>, to outer mitochondrial membrane in (<i>c</i>), and to septae in (<i>d</i>); <i>bars</i>, 0.2 µm.</p

Reducing the hydrophobicity of the first transmembrane segment of yeast Atp9p improves its import into mitochondria.

<p>The <i>Δatp9</i> strain was transformed with a hybrid Atp9 gene (Atp9-Hyb) encoding the mitochondrial targeting sequence (MTS) and first transmembrane segment (TMH1) of the <i>PaAtp9-7</i> protein, followed by the connecting loop and second transmembrane segment (TMH2) of yeast Atp9p (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen-1002876-g002" target="_blank">Figure 2A</a> for amino acid sequence and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s004" target="_blank">Figure S3E</a> for nucleotide sequence). Total cellular (<i>T</i>), mitochondrial (<i>M</i>) and post-mitochondrial supernatant (<i>C</i>) protein extracts were prepared from <i>WT</i> and <i>Δatp9</i>+Atp9-Hyb strains. Samples were separated by SDS-PAGE and probed with antibodies against yeast Atp9p and the cytosolic protein Pgk1p (phosphoglycerate kinase).</p

The <i>P. anserina Atp9</i> genes restore respiratory competence in <i>Δatp9</i> yeast.

<p>The <i>Δatp9</i> strain was transformed with CEN or 2 µ plasmids harbouring <i>PaAtp9-7</i> or <i>PaAtp9-5</i>, yielding strains AMY8 (<i>Δatp9+PaAtp9-7, CEN</i>), AMY11 (<i>Δatp9+PaAtp9-7</i>, 2 µ), AMY7 (<i>Δatp9+PaAtp9-5</i>, CEN), and AMY10 (<i>Δatp9+PaAtp9-5</i>, 2 µ). A) Growth curves of all strains in rich glycerol/ethanol medium at 28°C (subsequent panels use the same growth conditions). B) ATP synthase levels in <i>WT</i> and AMY10 revealed by separation of isolated mitochondria by BN-PAGE. The ATP synthase complexes (<i>V₁</i>, monomer; <i>V_n</i>, oligomers) and free F₁ were revealed in-gel by their ATPase activity (right). C) Total cellular (<i>T</i>) and mitochondrial (<i>M</i>) protein extracts were prepared from <i>WT</i>, AMY7 and AMY10 grown in YPEG. Samples (20 µg) were separated by SDS-PAGE and probed with antibodies against yeast Atp6p and γ-F₁. D) Differential interference contrast microscopy (left, ‘Nomarski’) and DAPI staining/fluorescence microscopy (middle) of <i>WT</i> and AMY10 cells. Right (‘Ultrastructure’) are electron micrographs of AMY10 cells (80 nm-thin sections). <i>V</i>, vacuole; <i>n</i>, nucleus; <i>m</i>, mitochondria.</p

Respiratory and ATP hydrolysis/synthesis activities of mitochondria.

<p>Mitochondria were isolated from yeast cells grown at 28°C in rich glycerol+ethanol (YPEG) or rich galactose (YPGALA), as indicated. All cultures contained less than 5% ρ⁻/ρ⁰ cells, except that of <i>Δatp9</i> where about 50% ρ⁻/ρ⁰ cells were scored. Additions were 0.15 mg/ml proteins, 4 mM NADH, 150 mM ADP, 4 mM CCCP, and 3 µg/ml oligomycin (<i>Oligo</i>). The values reported are averages of triplicate assays ± standard deviation. Respiratory and ATP synthesis activities were measured on freshly isolated, osmotically-protected mitochondria buffered at pH 6.8. For the ATPase assays, mitochondria kept at −80°C were thawed and the reaction was performed in absence of osmotic protection at pH 8.4. <i>ND</i>, not determined.</p

Genotypes and sources of yeast strains.

<p>Genotypes and sources of yeast strains.</p