The putative helical lid of the Hsp70 peptide-binding domain is required for efficient preprotein translocation into mitochondria.
The mitochondrial Hsp70 (Ssc1) is an essential component of the preprotein import machinery, responsible for the unfolding and movement of polypeptide chains through the mitochondrial membranes into the matrix. Here, we have analyzed the role of the carboxy-terminal variable domain during the protein translocation reaction. This segment is thought to form an alpha-helical lid over the substrate binding site. Truncated Ssc1 molecules lacking parts or all of the lid region showed reduced binding to substrate proteins but were able to interact with the co-chaperone Mge1 and the inner membrane anchor Tim44. Deletions of the complete lid resulted in a lethal phenotype in vivo, caused by the inability to sustain a productive preprotein import function. The translocation defect in vitro was not overcome by artificial unfolding of the preprotein prior to the import reaction. Despite a reduced substrate affinity, the presence of a minimal lid segment in Ssc1 was sufficient to support preprotein import. However, at low reaction temperatures or low matrix ATP levels, protein import rates were significantly reduced due to an unproductive interaction with the preprotein in transit. We conclude that the carboxy-terminal domain performs a crucial role in the import process by enhancing the import motor function of Ssc1 during polypeptide translocation.
Strub
Andreas
A
Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
Zufall
Nicole
N
Voos
Wolfgang
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003080:Cold Temperature; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D013379:Substrate Specificity
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate