The most abundant mitochondrial homolog of Hsp70, Ssc1p, is involved in the import and folding of mitochondrial proteins. We have developed an easy and efficient method for purifying Ssc1p. Following a first step of anion exchange at pH 6.6, a column of Mge1(His)(6) immobilized on Ni(2+)-agarose provides an efficient second dimension that results in highly purified protein. The strong and specific interaction between Ssc1p and its cofactor protein, Mge1, ensures that primarily functional protein is isolated. Ssc1p purified by this method hydrolyzed ATP with a turnover rate of 0.3/min. The ATP hydrolysis was enhanced slightly by Mge1, about 5 times by Mdj1, and 12 times by both cofactors together. The CD spectrum of Ssc1p had a pattern and temperature dependence similar to those shown for other hsp70 homologs, with a midpoint of the major transition at approximately 70 degrees C.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1006/prep.2001.1563 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase.
J Biol Chem
August 2002
Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 80-822 Gdansk, Kladki 24, Poland.
Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system.
View Article and Find Full Text PDFTwo-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His)(6) immobilized on Ni-agarose.
Protein Expr Purif
March 2002
George Wise Faculty of Sciences, Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel.
The most abundant mitochondrial homolog of Hsp70, Ssc1p, is involved in the import and folding of mitochondrial proteins. We have developed an easy and efficient method for purifying Ssc1p. Following a first step of anion exchange at pH 6.
View Article and Find Full Text PDFImportance of two ATP-binding sites for oligomerization, ATPase activity and chaperone function of mitochondrial Hsp78 protein.
J Mol Biol
December 2001
Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Kladki 24, Gdansk, 80-822, Poland.
The yeast mitochondrial chaperone Hsp78, a homologue of yeast cytosolic Hsp104 and bacterial ClpB, is required for maintenance of mitochondrial functions under heat stress. Here, Hsp78 was purified to homogeneity and shown to form a homo-hexameric complex, with an apparent molecular mass of approximately 440 kDa, in an ATP-dependent manner. Analysis of its ATPase activity reveals that the observed positive cooperativity effect depends both on Hsp78 and ATP concentration.
View Article and Find Full Text PDFLoss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells.
J Cell Sci
October 2001
Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates.
View Article and Find Full Text PDFMitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding.
FEBS Lett
January 2001
Department of Molecular and Cellular Biology, University of Gdansk, Poland.
The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!