8 research outputs found

    Loss of <i>hsp3102</i>, <i>hsp3104</i>, <i>hsp3105</i> and <i>sdj1</i> leads to delay of autophagy during stationary phase.

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    <p><i>S</i>. <i>pombe</i> cells expressing CFP-Atg8 were grown in YES medium and harvested at logarithmic phase (LP) (A) and prolonged stationary phase (SP) (B-D). (E) Deletion of <i>atg5</i> abolished CFP-Atg8 processing. Cells were lysed by sodium hydroxide and cell-free lysates were analyzed by immunoblotting using antibodies against GFP and Sla1 (loading control). The presence of free CFP is an indicator of autophagic flux.</p

    Western blot analysis of Hsp3101, Hsp3102, Hsp3105 and Sdj1 in wild-type, Δ<i>sty1</i> and Δ<i>atf1</i> cells.

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    <p>Wild-type, Δ<i>sty1</i> and Δ<i>atf1</i> cells were grown in YES medium. Cell lysates were prepared, and proteins were separated by SDS/PAGE and analyzed by immunoblotting with indicated antibodies. Sla1 serves as the loading control.</p

    Deletion of <i>hsp31</i> and <i>sdj1</i> genes affects subcellular localization of GFP-Atg8.

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    <p>(A) GFP-Atg8 localization in wild type and deletion mutants of <i>hsp31</i> and <i>sdj1</i>. Cells were grown in YES medium and analyzed by fluorescence microscopy. Samples were taken at indicated time points during growth. (B) Quantitative analysis of the GFP-Atg8 puncta in the panel (A) (mean ± SEM; n>500 cells). Statistical significance (*P < 0.05) was determined by Student's <i>t</i> test.</p

    Induction of <i>S</i>. <i>pombe</i> homologs of DJ-1.

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    <p>(A) qRT-PCR analysis of expression of <i>S</i>. <i>pombe hsp3101-hsp3105</i> and <i>sdj1</i> genes in the wild-type cells. Total RNA was isolated from the wild-type cells grown in YES medium at the indicated time points. All mRNA levels were normalized to the control <i>act1</i><sup>+</sup> mRNA level and were expressed as fold change relative to the mRNA levels at the 7 h time point, which was set at a value of 1. Data are presented as mean ± SD (<i>p</i> ≤0.01; <i>t</i> test). (B) Immunoblot analyses of Hsp3101, Hsp3102, Hsp3105 and Sdj1 expression in wild-type cells. Crude extracts were prepared from the wild-type cells at indicated time points (h). Total proteins were separated on SDS/PAGE gels and immunoblotted using anti-Hsp3101 Ab, anti- Sdj1 Ab, anti-Myc Ab, which detects Hsp3102-Myc and Hsp3105-Myc, and anti-Sla1 Ab (serves as a loading control).</p

    <i>Schizosaccharomyces pombe</i> Homologs of Human DJ-1 Are Stationary Phase-Associated Proteins That Are Involved in Autophagy and Oxidative Stress Resistance

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    <div><p>The Parkinson′s disease protein DJ-1 is involved in various cellular functions including detoxification of dicarbonyl compounds, autophagy and oxidative stress response. DJ-1 homologs are widely found in both prokaryotes and eukaryotes, constituting a superfamily of proteins that appear to be involved in stress response. <i>Schizosaccharomyces pombe</i> contains six DJ-1 homologs, designated Hsp3101-Hsp3105 and Sdj1 (previously named SpDJ-1). Here we show that deletion of any one of these six genes somehow affects autophagy during prolonged stationary phase. Furthermore, deletions of each of these DJ-1 homologs result in reduced stationary phase survival. Deletion of <i>sdj1</i> also increases the sensitivity of stationary-phase cells to oxidative stress induced by hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) whereas overexpression of <i>sdj1</i> has the opposite effect. Consistent with their role in stationary phase, expression of <i>hsp3101</i>, <i>hsp3102</i>, <i>hsp3105</i> and <i>sdj1</i>, and to a lesser extent <i>hsp3103</i> and <i>hsp3104</i>, is increased in stationary phase. The induction of <i>hsp3101</i>, <i>hsp3102</i>, <i>hsp3105</i> and <i>sdj1</i> involves the Sty1-regulated transcription factor Atf1 but not the transcription factor Pap1. Our results firmly establish that <i>S</i>. <i>pombe</i> homologs of DJ-1 are stationary-phase associated proteins and are likely involved in autophagy and antioxidant defense in stationary phase of <i>S</i>. <i>pombe</i> cells.</p></div

    Expression levels of <i>S</i>. <i>pombe DJ-1</i> homologs in wild-type, Δ<i>sty1</i>, Δ<i>atf1</i> and Δ<i>pap1</i> cells.

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    <p><i>S</i>. <i>pombe hsp3101</i>-<i>hsp3105</i> and <i>sdj1</i> in Δ<i>sty1</i> (A), Δ<i>atf1</i> (B) and Δ<i>pap1</i> (C) were grown in YES medium, and total RNAs were extracted at indicated times and analyzed by qRT-PCR. Columns indicate mean values of at least three independent experiments; error bars represent standard error of the mean (SEM) (n = 3, separate experiments). Statistical analyses were performed using Student <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01). Reliable data could not be obtained beyond the 48 h time point due to the loss of viability caused by <i>sty1 or atf1</i> depletion.</p

    Deletion of <i>S</i>. <i>pombe</i> DJ-1 homologs reduces cell survival in stationary phase.

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    <p>Cells were grown in low-glucose rich YEPD medium (1% glucose). The survival difference between the wild-type and mutants is more noticeable when cells were grown in YEPD. At the indicated time points, an aliquot of each culture was taken, serially diluted and plated to obtain viable colonies. Viability of stationary phase cells was measured as the ability of cells to form colonies on rich medium plates. The graph shows the percentage of viable cells ± SEM. The viability of cells in exponential growth (12 h) was set to 100%.</p
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