The J domain–related cochaperone Tim16 is a constituent of the mitochondrial TIM23 preprotein translocase

Abstract

Mitochondria import the vast majority of their proteins from the cytosol. The mitochondrial import motor of the TIM23 translocase drives the translocation of precursor proteins across the outer and inner membrane in an ATP-dependent reaction. Tim44 at the inner face of the translocation pore recruits the chaperone mtHsp70, which binds the incoming precursor protein. This reaction is assisted by the cochaperones Tim14 and Mge1. We have identified a novel essential cochaperone, Tim16. It is related to J-domain proteins and forms a stable subcomplex with the J protein Tim14. Depletion of Tim16 has a marked effect on protein import into the mitochondrial matrix, impairs the interaction of Tim14 with the TIM23 complex and leads to severe structural changes of the import motor. In conclusion, Tim16 is a constituent of the TIM23 preprotein translocase, where it exerts crucial functions in the import motor.

OWL1: An Arabidopsis J-Domain Protein Involved in Perception of Very Low Light Fluences

“…In other cases, the Hsp70 and type III J-domain protein are recruited independently to the site of action, where the J-domain protein stimulates ATP hydrolysis by the partner Hsp70. This can be observed for the import machinery of mitochondria and chloroplasts (Westermann and Neupert, 1997;Becker et al, 2004;Kozany et al, 2004;Qbadou et al, 2007). The divergence of the C terminus among the type III J-domain proteins and their uniqueness suggests the possibility that they bind very different proteins.…”

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

“…However, beside the translocase-associated portion of the cochaperones Pam16 and Pam18, a significant non-translocase pool of the proteins has been found in mitochondria 11,26,30,31,35 . This, situation is now explained by the observed dynamics of the active translocase.…”

“…3b, the mutant form of Pam18 displays faster migration in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and is efficiently recognized by the anti-Pam18 antiserum, lane 1 versus 2). Interestingly, despite the fact that Pam18 and Pam16 are considered to form a stable complex 11,16,26,31 , the inhibitory Pam16 remained translocase-associated in pam18-66 mitochondria. Thus, we concluded that the observed loss of motor function and concomitant instability of the translocation intermediate could be attributed to defective ATPase cycling at the import motor.…”

“…4e). It should be noted that although this assay only allows to analyse the integration of subunits, we assume that a release occurs to a similar extent as indicated by the presence of a free pool of Pam16 and Pam18 in mitochondria 11,26,30,31,35 . Thus, our experiments demonstrated that Tim21 and subunits of the PAM complex can be recruited into a precursor-occupied and active translocase, a state that requires import motor activity for formation and maintenance.…”

“…For both activities, the soluble co-chaperone Mge1 stimulates ADP/ATP exchange 9,10 . Although matrix co-chaperones act as J-proteins stimulating the ATPase activity of mtHsp70 during protein folding, a TIM23 complex-associated membrane integral J-protein (Pam18) is import specific [11][12][13][14][15] . At the resting translocase, Pam18 forms a complex with the J-like Pam16.…”