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Analysis of Complex Interactions Between the Essential Subunits, Pam16, Tim44, and Tim50, of the Hsp70‐based Mitochondrial Protein Import Machinery
Author(s) -
Yan Nicholas Lok,
Ting SeeYeun,
Schilke Brenda,
Craig Elizabeth A
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.760.9
Subject(s) - mitochondrial matrix , microbiology and biotechnology , biology , chaperone (clinical) , cytosol , mitochondrion , translocon , inner mitochondrial membrane , immunoprecipitation , biophysics , biochemistry , membrane protein , membrane , gene , medicine , pathology , enzyme
Because the vast majority of mitochondrial proteins are synthesized in the cytosol, efficient protein import is essential for mitochondrial function and cell viability. The mitochondrial protein import motor, which is tethered to the matrix face of the inner membrane, plays a critical role. This motor translocates preproteins from the cytosol, across both mitochondrial membranes, into the mitochondrial matrix. This biological machine is driven by the molecular chaperone mtHsp70, which binds translocating preproteins entering the matrix in an ATP‐dependent manner. Both mtHsp70 and its co‐chaperone Pam18, which performs the critical function of stimulating Hsp70's ATPase activity, are tethered to the translocon by Tim44. While mtHsp70 directly interacts with Tim44, evidence suggests that Pam18 is tethered via Pam16, with which it forms a heterodimer. However, how Pam16 interacts with Tim44 is not well understood. This study aims to determine how Pam16 interacts with Tim44 and the functional consequences of the interaction. Our previous genetic and co‐immunoprecipitation analyses implicated distinct regions in Pam16 and Tim44, both in their N‐termini, that may be the sites of interaction. Alterations in these regions not only caused Pam16 to dissociate from the translocon, but also led to temperature‐sensitive growth defects, demonstrating the importance of this interaction. We now report site‐specific crosslinking demonstrating that Pam16 and Tim44 are in close proximity to each other. When a photoactivatable crosslinker was incorporated at several positions in these regions, Pam16 could be crosslinked to Tim44, or vice versa. Altogether, these results suggest that Pam16 directly interacts with Tim44 via these regions. In addition to crosslinking to Tim44, we found that the extreme N terminus of Pam16 also crosslinked to Tim50, an essential translocon subunit that facilitates movement of the preprotein from the translocon of the outer membrane to that of the inner membrane. Tim50 is a transmembrane protein with an essential intermembrane space domain and a matrix‐exposed region whose function is unknown. Taken together, these results support the idea of Pam16 as an essential tether in the import motor, mediating complex interactions with subunits that are necessary for mitochondrial protein import.