Pam17 Is Required for Architecture and Translocation Activity of the Mitochondrial Protein Import Motor

Abstract

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preproteinbinding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.Mitochondria possess about 900 different proteins in yeast and about 1,500 different proteins in humans (34,42,44). Ninety-nine percent of the proteins are synthesized on cytosolic ribosomes. Studies of the past years revealed the existence of multiple protein import pathways into the four mitochondrial subcompartments: outer membrane, intermembrane space, inner membrane, and matrix (8,15,16,18,20,29,33). A major pathway is used by preproteins carrying the classical amino-terminal presequences that direct translocation into the matrix and are removed by the mitochondrial processing peptidase. These cleavable preproteins use the general translocase of the outer membrane (TOM complex), which contains surface receptors and the import channel Tom40, and then engage the specific presequence translocase of the inner membrane (TIM23 complex). The membrane potential ⌬ across the inner membrane drives the translocation of the presequences through the TIM23 complex. Some cleavable preproteins contain a hydrophobic stop-transfer signal behind the positively charged presequence (13). Those preproteins are laterally released from the TIM23 complex and are thus sorted into the inner membrane. Most preproteins with a presequence, however, do not carry hydrophobic sorting signals and are thus completely translocated into the mitochondrial matrix. This transport strictly requires the presequence translocase-associated motor (PAM) (26,36).The presequence translocase contains four different integral proteins of the inner mitochondrial membrane (4,10,18,30,36,48): the channel-forming protein Tim23; Tim50 that cooperates with Tim23 in binding of preproteins on the intermembrane space side; Tim17 that plays a dual role in promoting protein sorting into the inner membrane and binding of PAM subunits to the translocase; and Tim21 that transiently connects...

Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

“…mtHsp70 is recruited to the translocase by Tim44 and binds to the incoming precursors in an ATP-dependent manner that is regulated by nucleotide exchange factor Mge1 and the J-complex Tim14-Tim16. In addition to these eight subunits essential for cell viability and function of the translocase, the TIM23 complex contains two recently identified nonessential subunits, Tim21 and Pam17 Mokranjac et al, 2005;van der Laan et al, 2005), which appear to modulate the activity of the translocase in an antagonistic manner .…”

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

“…Pam18 inactivation after precursor accumulation led to a similar loss of the TOM/TIM23 supercomplex, as observed in the ssc1-3 mutant. Although previous BN-PAGE analyses indicated that mtHsp70/Tim44 were not a stoichiometric constituents of the TOM/TIM23 supercomplex 25 , subsequent analyses showed that all constituents of the import motor are recovered with the TOM/ TIM23 supercomplex when mild affinity isolations are employed 13,[26][27][28][29][30] . Accordingly, at the active translocase, ATP hydrolysis by Hsp70, stimulated by its co-chaperone Pam18, was required to sustain the matrix-destined translocation intermediate.…”

“…In contrast to mutants of the co-chaperones, pam17D and mgr2D cells are viable, but are characterized by growth retardation 28,32 . Nevertheless, pam17D and mgr2D mitochondria displayed selective matrix protein import defects (Fig.…”

“…Import motor defects affect matrix protein import and translocation of tightly folded domains 23,28 . In contrast to mutants of the co-chaperones, pam17D and mgr2D cells are viable, but are characterized by growth retardation 28,32 .…”

Mode of membrane insertion of individual transmembrane segments in Mdl1 and Mdl2, multi‐spanning mitochondrial ABC transporters

“…The PAM complex, consisting of Pam16p, Pam17p, Pam18p, mtHsp70, Mge1p and Tim44p, modulates the translocation of proteins into the matrix [4,28,35,36]. Therefore, impaired PAM machinery function leads to defects in the import of matrix-targeted proteins [28].…”