The Pam18/Tim14-Pam16/Tim16 complex of the mitochondrial translocation motor: the formation of a stable complex from marginally stable proteins.
The vast majority of mitochondrial proteins are imported from the cytosol. For matrix-localized proteins, the final step of translocation across the inner membrane is mediated by the mitochondrial translocation motor, of which mhsp70 is a key component. The ATP-dependent function of mhsp70 is regulated by a complex, composed of a J-protein (called Pam18 or Tim14) and a J-like protein (called Pam16 or Tim16), and the nucleotide exchange factor Mge1. In this study, we investigated the structural properties of a recombinant purified Pam18/Tim14-Pam16/Tim16 complex using cross-linking with the bifunctional reagent DSS and CD-spectroscopy. The results of the study show that both Pam18/Tim14 and Pam16/Tim16 are thermally unstable proteins that unfold at very low temperatures (T(m) values of 16.5 degrees C and 29 degrees C, respectively). Upon mixing the proteins in vitro, or when both proteins are co-overexpressed in bacteria, Pam18/Tim14 and Pam16/Tim16 form a heterodimer that is thermally more stable than the individual proteins (T(m) = 41 degrees C). Analysis of the properties of the complex in GdnHCl shows that dissociation of the heterodimer is the limiting step in achieving full denaturation.
Iosefson
Ohad
O
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69778, Israel.
Levy
Ran
R
Marom
Milit
M
Slutsky-Leiderman
Olga
O
Azem
Abdussalam
A
D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C480348:PAM18 J protein, S cerevisiae; C484526:Pam16 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins