A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.
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 (Delta(1)-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 (Schülke, 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.
Schülke
N
N
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Sepuri
N B
NB
Gordon
D M
DM
Saxena
S
S
Dancis
A
A
Pain
D
D
D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D011487:Protein Conformation; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D011762:Pyrroline Carboxylate Reductases; D005982:Glutathione Transferase