Mutations in a Putative Zinc-Binding Domain Inactivate the Mitochondrial Intermediate Peptidase

The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/G)XXXX(↓), from the N-terminus of many imported mitochondrial proteins. This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of m...

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Published inBiochemical and biophysical research communications Vol. 226; no. 3; pp. 822 - 829
Main Authors Chew, Anne, Rollins, Robert A., Sakati, Wayne R., Isaya, Grazia
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 24.09.1996
Subjects
Amino Acid Sequence
Binding Sites
Cloning, Molecular
Codon
Conserved Sequence
Cysteine
DNA Mutational Analysis
Enzyme Stability
Escherichia coli
Ethylmaleimide - pharmacology
Iodoacetates - pharmacology
Iodoacetic Acid
Metalloendopeptidases - biosynthesis
Metalloendopeptidases - chemistry
Metalloendopeptidases - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Oxygen Consumption
Protein Precursors - metabolism
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - metabolism
Restriction Mapping
Saccharomyces cerevisiae - growth & development
Saccharomyces cerevisiae - metabolism
Zinc Fingers
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Summary:The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/G)XXXX(↓), from the N-terminus of many imported mitochondrial proteins. This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of metallo- and cysteine-proteases, respectively. To elucidate the mechanism of action of MIP, we analyzed by site-directed mutagenesis the functional role of a putative zinc-binding domain (F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E) and two cysteine residues (C131 and C581), which are highly conserved in evolutionarily distant MIP sequences. We show that two histidines and a glutamic acid in the H-E-X-G-H motif and a glutamic acid 25 residues from the second histidine are essential for MIP functionin vivo.In contrast, C131 and C581 are important for protein stability but are not required for activityin vivoorin vitro.These findings are consistent with MIP being a metallopeptidase.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1996.1435