The interplay between components of the mitochondrial protein translocation motor studied using purified components.
The final step of protein translocation across the mitochondrial inner membrane is mediated by a translocation motor composed of 1) the matrix-localized, ATP-hydrolyzing, 70-kDa heat shock protein mHsp70; 2) its anchor to the import channel, Tim44; 3) the nucleotide exchange factor Mge1; and 4) a J-domain-containing complex of co-chaperones, Tim14/Pam18-Tim16/Pam16. Despite its essential role in the biogenesis of mitochondria, the mechanism by which the translocation motor functions is still largely unknown. The goal of this work was to carry out a structure-function analysis of the mitochondrial translocation motor utilizing purified components, with an emphasis on the formation of the Tim44-mHsp70 complex. To this end, we purified Tim44 and monitored its interaction with other components of the motor using cross-linking with bifunctional reagents. The effects of nucleotides, the J-domain-containing components, and the P5 peptide (CALLSAPRR, representing part of the mitochondrial targeting signal of aspartate aminotransferase) on the formation of the translocation motor were examined. Our results show that only the peptide and nucleotides, but not J-domain-containing proteins, affect the Tim44-mHsp70 interaction. Additionally, binding of Tim44 to mHsp70 prevents the formation of a complex between the latter and Tim14/Pam18-Tim16/Pam16. Thus, mutually exclusive interactions between various components of the motor with mHsp70 regulate its functional cycle. The results are discussed in light of known models for the function of the mitochondrial translocation motor.
Slutsky-Leiderman
Olga
O
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69778, Israel.
Marom
Milit
M
Iosefson
Ohad
O
Levy
Ran
R
Maoz
Sharon
S
Azem
Abdussalam
A
D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008954:Models, Biological; D018832:Molecular Chaperones; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C086039:MGE1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D009711:Nucleotides; C484526:Pam16 protein, S cerevisiae; D010455:Peptides; D011994:Recombinant Proteins; C090869:SSA3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C077016:TIM44 protein, S cerevisiae; C478853:Tim14 protein, S cerevisiae