A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates

Norbert Schülke, Naresh Babu V. Sepuri, Donna M. Gordon, Sandeep Saxena, Andrew Dancis, Debkumar Pain

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39 Scopus citations

Abstract

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Δ1-pyrroline-5- carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively 'zippered' together, forming long stretches in which they are in close contact (Schulke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.

Original languageEnglish (US)
Pages (from-to)22847-22854
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number32
DOIs
StatePublished - Aug 6 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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