Pam16 has an essential role in the mitochondrial protein import motor

Abstract

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.

Taz1, an Outer Mitochondrial Membrane Protein, Affects Stability and Assembly of Inner Membrane Protein Complexes: Implications for Barth Syndrome

“…In vitro import into isolated yeast mitochondria (performed at 30°C), protease treatment of mitochondria, and carbonate extraction have been described previously Truscott et al, 2003;Frazier et al, 2004). Standard techniques were used for SDS-PAGE and Western blotting.…”

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.…”

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

“…Therefore, impaired PAM machinery function leads to defects in the import of matrix-targeted proteins [28]. In case of Mgm1p, defective PAM function impairs the import of Mgm1p; therefore, the formation of the sMgm1p isoform was reduced [37] (Fig.…”

“…Yeast transformants of W303-1a (MATa, ade2, can1, his3, leu2, trp1, ura3), W303-1a rho À [26], Dyta10 (MATa, ade2-1, his3-11,15, yta10::HIS3MX6, trp1-1, leu2,112, ura3-52) [27], the temperature-sensitive pam16-3 mutant or the isogenic wild-type PAM16 strain [28] carrying each plasmid were grown overnight in 5 ml of -Leu medium at 30°C. The preparation of whole-cell lysates by trichloroacetic acid (TCA) precipitation, SDS-PAGE and Western blotting were followed as described in [18].…”

“…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].…”