Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.
Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.
Vasiljev
Andreja
A
Institut für Physiologische Chemie der Universität München, D-81377 Munich, Germany.
Ahting
Uwe
U
Nargang
Frank E
FE
Go
Nancy E
NE
Habib
Shukry J
SJ
Kozany
Christian
C
Panneels
Valérie
V
Sinning
Irmgard
I
Prokisch
Holger
H
Neupert
Walter
W
Nussberger
Stephan
S
Rapaport
Doron
D
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017385:Sequence Homology
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C107250:TIM10 protein, S cerevisiae; C102549:TIM22 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae