Yvh1 protein phosphatase is required for pre-autophagosomal structure formation after TORC1 inactivation

<p>The pre-autophagosomal structure (PAS) is a putative site for autophagosome formation in budding yeast. Upon nutrient depletion or rapamycin treatment, target of rapamycin complex 1 (TORC1) becomes inactive, inducing PAS formation and autophagy. Here, we show that Yvh1 phosphatase is critical for PAS formation, but not autophagy induction, after TORC1 inactivation.</p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -5

Matched in the w>database search indicated with asterisks. Panels correspond to a spot from the 20S() and 20S() proteasomes, respectively.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -2

20S proteasome, and 10 μM of peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) at 37°C for 1 h. Reactions were started and stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD for three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -10

Ome, 1 mM EDTA-Tris, pH 8.5, 10% DMSO, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of linolenic acid at 37°C for 1 h. The reactions were started, stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -0

50 mM Tris-HCl, pH 7.6, 1 μg/ml 20S proteasome, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of SDS at 37°C for 1 h. The reactions were started and stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -1

Some, 1 mM EDTA-Tris, pH 8.5, 10% DMSO, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of linolenic acid at 37°C for 1 h. The reactions were started, stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -7

Erred to a PVDF membrane. The α1- and α7-subunits were detected using their respective antibodies as described in the methods. () Cells from wild-type and the mutant (wt, ) grown at 25°C (OD= 1.0) were harvested and the cell extracts were applied to a Q-sepharose column. Active Q-sepharose fractions were concentrated and treated with (AP+) or without (AP-) alkaline phosphatase as described in the methods. The samples were then subjected to SDS-PAGE in a 12% polyacrylamide gel. The α7-subunit was detected by immunostaining with anti-α7 antibody [,].<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -8

Ltures were then immediately shifted to 37°C and incubated for several hours as indicated. Cell extracts were applied to a Q-sepharose column. The molecular weight level of the α7-subunits in both strains produced during the indicated incubation period was assayed by Western blotting using anti-α7 antibody.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, -9

50 mM Tris-HCl, pH 7.6, 1 μg/ml 20S proteasome, and 10 μM peptidyl substrates (: Suc-LLVY-MCA for chymotrypsin-like activity; : Z-LLE-MCA for PGPH activity; : Boc-LRR-MCA for trypsin-like activity) and increasing concentrations of SDS at 37°C for 1 h. The reactions were started and stopped as described in methods. Symbols represent the wild-type strain (○) and the mutant (●). Values are means ± SD of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, "</p><p>http://www.biomedcentral.com/1471-2091/9/20</p><p>BMC Biochemistry 2008;9():20-20.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2515314.</p><p></p

Elucidation of novel budding yeast separase mutants

<p>The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase <i>ESP1</i> in the budding yeast <i>Saccharomyces cerevisiae</i>. All mutants were defective in sister chromatid separation at the restricted temperature. Some <i>esp1</i>-<i>ts</i> mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some <i>esp1</i>-<i>ts</i> mutants, whereas it rather exacerbated it in others. The <i>Drosophila</i> Pumilio homolog <i>MPT5</i> was isolated as a high-dosage suppressor of <i>esp1</i>-<i>ts</i> cells. We discuss various features of separase based on these findings.</p> <p>Securin positively and negatively regulates separase function. In addition, extracellular inputs affect separase function.</p