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(decreases), 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 function in vivo. In contrast, C131 and C581 are important for protein stability but are not required for activity in vivo or in vitro. These findings are consistent with MIP being a metallopeptidase.
Chew
A
A
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Rollins
R A
RA
Sakati
W R
WR
Isaya
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon; D017124:Conserved Sequence; D003545:Cysteine; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D004926:Escherichia coli; D005033:Ethylmaleimide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010101:Oxygen Consumption; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D016335:Zinc Fingers
D003062:Codon; D007461:Iodoacetates; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; C068038:mitochondrial intermediate peptidase; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid