Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor.
The heat-shock protein 70 (Hsp70)-based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
June E
JE
Pais
Brenda
B
Schilke
Elizabeth A
EA
Craig
D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009154:Mutation; D055503:Protein Multimerization / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; 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; C090869:SSA3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins