Over-expression, purification and determination of the proteolytic processing site of the yeast mitochondrial CBS1 protein.
Yeast transformants harboring the CBS1 gene under the control of the strong ADC1 promoter on a high copy number plasmid express the mitochondrial CBS1 protein at artificially high levels. Over-expressed protein is imported into mitochondria and correctly processed to yield the mature mitochondrial 23.5 kDa form, but differs in its solubility properties from CBS1 in wild-type mitochondria. It forms insoluble protein aggregates, which are refractory to solubilization with 1% Taurodeoxycholate. We exploited this observation to separate CBS1 from the bulk of mitochondrial proteins and to isolate CBS1 after SDS gel electrophoresis. Determination of the amino-terminal amino acids of the purified protein reveals that the mature CBS1 protein starts with Ile30, at the characteristic distance of +2 amino acids from an arginine residue (Arg28). The cleavage site shows a remarkable homology to that of subunit 9 of the F0F1 ATPase from Neurospora crassa.
Körte
A
A
Lehrstuhl für Genetik, Universität München, Federal Republic of Germany.
Michaelis
U
U
Lottspeich
F
F
Rödel
G
G
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013482:Superoxide Dismutase / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D015534:Trans-Activators; D014170:Transformation, Genetic
C060724:CBS1 protein, S cerevisiae; D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D013912:Threonine; D001120:Arginine; D013482:Superoxide Dismutase; D006180:Proton-Translocating ATPases