Mutations in a putative zinc-binding domain inactivate the mitochondrial intermediate peptidase

Anne Chew, Robert A. Rollins, Wayne R. Sakati, Grazia Isaya

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Abstract

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 lender 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.

Original languageEnglish (US)
Pages (from-to)822-829
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume226
Issue number3
DOIs
StatePublished - Sep 24 1996

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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