Lactic dehydrogenase and cytochrome <i>b</i>2 of baker's yeast. Purification and crystallization

Appleby, Cyril A.; Morton, R. K.

doi:10.1042/bj0710492

Cited by 186 publications

(68 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Previously published procedures were used to assay fumarase (48), using an extinction coefficient of 2.44 (4); glucose-6-phosphate dehydrogenase (19); cytochrome c oxidase (42); cytochrome b2 (as lactate dehydrogenase [1]); PSC dehydrogenase (13); and 3-galactosidase (44). Protein concentrations were determined by the method of Bradford (11), with crystalline bovine serum albumin used as the standard.…”

Section: Methodsmentioning

confidence: 99%

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

Brandriss¹,

Krzywicki²

1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

Al-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible I-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of I-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of ,-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver I-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid ,-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

show abstract

Section: Methodsmentioning

confidence: 99%

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

Brandriss¹,

Krzywicki²

1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…The mitochondrial suspension was divided into aliquots and diluted 40-fold either in SEM buffer or in 10 mM HEPES (pH 7.4) and incubated for 15 min at O°C. Afterwards, each reaction was adjusted to 250 mM sucrose and 150 mM KCI and was divided into aliquots corresponding to 40 Fg of mitochondrial protein for treatment with PK (15 pg/ml final concentration) and for enzymatic measurement of cytochrome bp activity (Appleby and Morton, 1959).…”

Section: Subfractionationmentioning

confidence: 99%

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space

Koll

Guiard

Rassow

et al. 1992

Cell

219

151

View full text Add to dashboard Cite

“…The following procedures were performed according to published methods: enzyme assays of cytochrome b2 (Appleby and Morton, 1959), adenylate kinase, and fumarase (Schmidt et al, 1984); immunoprecipitation of cytochrome b2, cytochrome ~1, and the Fe/S protein (Schleyer et al, 1982;Hart1 et al, 1988); protein determination (Bradford, 1976); electrophoresis on 10% (cytochrome b2) and 17% (cytochrome c, and Fe/S protein) polyacrylamide gels (Laemmli, 1970); fluorography and quantitation of fluorographs by densitometry (Hart1 et al, 1986). reading the manuscript.…”

Section: Miscellaneousmentioning

confidence: 99%

Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide

Hartl¹,

Ostermann²,

Guiard

et al. 1987

Cell

270

143

View full text Add to dashboard Cite

SummaryWe investigated the import and sorting pathways of cytochrome b2 and cytochrome cl, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediatesized forms, which art? subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sizedforms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.

show abstract

Lactic dehydrogenase and cytochrome b2 of baker's yeast. Purification and crystallization

Cited by 186 publications

References 21 publications

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space

Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide

Contact Info

Product

Resources

About