The Presequence Translocase-associated Protein Import Motor of Mitochondria

Abstract

Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM). The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16. Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif HisPro-Asp (HPD). We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18. Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70. The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity. Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18. Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria.Mitochondria import hundreds of different precursor proteins that are synthesized in the cytosol (1-6). A large class of precursor proteins carries amino-terminal-cleavable targeting signals (presequences) that direct these preproteins across both mitochondrial membranes into the matrix. The preproteins are recognized by receptors of the translocase of the outer membrane and transported across the outer membrane by the general import pore. After traversing the intermembrane space, the preproteins are recognized by the presequence translocase of the inner membrane (TIM23 complex). 1 The presequence translocase forms a voltage-activated translocation channel across the inner membrane through which the loosely folded precursor polypeptides pass (7). The membrane potential across the inner membrane, however, only promotes the translocation of the positively charged amino-terminal presequences across the inner membrane.The import of the major portion of the precursor polypeptide chain into the matrix requires the function of the presequence translocase-associated motor (PAM) (8 -11). The core of the PAM complex is formed by the mitochondrial heat shock protein of 70 kDa (mtHsp70, also termed Ssc1 in yeast). mtHsp70 drives the translocation and the unfolding of the preprotein by an ATP-dependent reaction (12, 13). Recent studies reveal that the PAM complex is one of the most complex Hsp70 systems (14). Besides the ATP-utilizing core component mtHsp70, which is essential for cell viability, four other essential cochaperones are needed for the motor function of PAM. The membrane protein Tim44 is associated with the TIM23 complex and stably interacts with mtHsp70 in a nucleotide-sensitive manner (15-17). The mitochondrial GrpE (Mge1) promotes the release of nucleotides from mtHsp70 and, thus, allows the completion of the mtHsp70 reaction cycle (18 -20). Most Hsp70s cooperate with coc...

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

“…Previous analyses indicated that Pam16 and Pam18 could form a heterodimer (18,19). Consistent with these findings, we found that upon mixing equimolar amounts of full-length Pam16 and Pam18, Ͼ75% of Pam16 was coimmunoprecipitated with antibodies specific for Pam18 (Fig.…”

“…Pam16 is not capable of stimulating Ssc1's ATPase activity and thus, is not a functional J protein partner of Ssc1 (19). However, whether the absence of this activity is an important aspect of Pam16's function is not known.…”

“…19 and data not shown). Therefore, we replaced Pam16's J-like domain with Pam18's functional J domain, forming a chimeric protein, Pam16:Pam18, composed of the N-terminal 52 aa of Pam16 and the J domain of Pam18 (amino acid s106-168; Fig.…”

“…In addition, the C-terminal region of Pam16 has significant sequence similarity to Pam18's C-terminal J domain. However, Pam16 lacks the critical HPD motif, having DKE in its place, and does not stimulate Ssc1's ATPase activity (19). Pam16 and Pam18 form a complex that has a reduced ability to stimulate Ssc1's ATPase activity compared to Pam18 alone.…”

“…Pam16 and Pam18 form a complex that has a reduced ability to stimulate Ssc1's ATPase activity compared to Pam18 alone. This inhibition has been suggested to play an important role in protein translocation (19,20).…”