Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

Abstract

Translocation of proteins across the mitochondrial inner membrane is an essential process requiring an import motor having mitochondrial Hsp70 (mtHsp70) at its core. The J protein partner of mtHsp70, Pam18, is an integral part of this motor, serving to stimulate the ATPase activity of mtHsp70. Pam16, an essential protein having an inactive J domain that is unable to stimulate mtHsp70's ATPase activity, forms a heterodimer with Pam18, but its function is unknown. We set out to test the importance of three properties of Pam16: (i) a stable interaction between Pam16 and Pam18, (ii) the inability of Pam16's degenerate J domain to stimulate Ssc1's ATPase domain, and (iii) the innately lower stimulatory activity of the Pam16:Pam18 heterodimer, compared to Pam18 alone. Neither substantial reduction in the ability of Pam18 to stimulate Ssc1's ATPase activity, nor the presence of an active J domain in Pam16, had deleterious effects on cell growth, indicating the lack of importance of two of these biochemical properties. However, a stable interaction between Pam16's degenerate J domain and Pam18's J domain was found to be critical for function. Alterations that destabilized the Pam16:Pam18 heterodimer had deleterious effects on cell growth and mitochondrial protein import; intragenic suppressors that restored robust growth also restored heterodimer stability. Our results support the idea that Pam16's J-like domain strongly interacts with Pam18's J domain, leading to a productive interaction of Pam18 with mtHsp70 at the import channel. mitochondria ͉ translocation ͉ Hsp40 ͉ Pam18 ͉ heterodimer

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase

“…Disruption of this heterodimeric subcomplex has been associated with loss of function and cellular lethality (20,38). The contrasting functions of both the J-proteins paralogs, however, do not reflect on their properties at the translocase channel (Fig.…”

“…The mitochondrial inner membrane J-proteins are thought to function in association with their corresponding J-like protein counterparts (18,38,43). Disruption of this heterodimeric subcomplex has been associated with loss of function and cellular lethality (20,38).…”

“…However, relatively stable interactions between the J-proteins dimers are well maintained. In contrast, a mild detergent treatment such as digitonin dissolves the intervening membranes without affecting the association of the translocase components (19,20,27,38). The isolated complexes can be resolved through blue native-PAGE and detected by immunoblotting.…”

Remodelling of the active presequence translocase drives motor-dependent mitochondrial protein translocation

“…A cycle of co-chaperone recruitment and subsequent release would allow coupling of the ATPase cycle of mtHsp70 to the activity of the translocon. At the resting translocase, Pam18 and Pam16 form a complex in which the J-protein is incapable of mtHsp70 activation 12,16,18 . Our finding that the recruitment of Pam18 into the active translocase is more efficient than that of Pam16 and that in case of the mgr2D mutant both proteins display a different mode of translocase recruitment (Figs 3h and 4b) suggests a model in which the J-protein is recruited as a free protein and concomitantly becomes inactivated by associating with Pam16 at the translocase.…”

“…The translocase-associated Tim44 is thought to position mtHsp70 at the trans side of the Tim23 channel (Fig. 1a) 12,18,19 . Thus, four essential co-chaperones are required at the translocase to regulate mtHsp70 function during protein import 20,21 .…”