A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria - PubMed (original) (raw)

. 2003 Nov 24;163(4):707-13.

doi: 10.1083/jcb.200308004.

Wolfgang Voos, Ann E Frazier, Maria Lind, Yanfeng Li, Andreas Geissler, Jan Dudek, Hanne Müller, Albert Sickmann, Helmut E Meyer, Chris Meisinger, Bernard Guiard, Peter Rehling, Nikolaus Pfanner

Affiliations

A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria

Kaye N Truscott et al. J Cell Biol. 2003.

Abstract

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

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Figures

Figure 1.

Figure 1.

Identification of an essential J-protein at the mitochondrial presequence translocase. (A) Purification of the presequence translocase from yeast mitochondria. Mitochondria from wild-type (WT) cells and cells expressing protein A–tagged Tim23 were lysed in digitonin and subjected to IgG chromatography. The TIM23 complex was eluted by TEV protease, separated by SDS-PAGE, and stained with colloidal Coomassie. The asterisk indicates a minor fraction of the abundant inner membrane protein ADP/ATP carrier (Geissler et al., 2002). 18K indicates the newly identified J-protein of 18 kD. (B) Deduced primary structure of the essential 18-kD J-protein of PAM. Gray box, hydrophobic segment; dashed line, J-domain. (C) Cellular fractionation. Wild-type spheroplasts were lysed and subjected to successive differential centrifugation steps. Equal volumes of the fractions were analyzed by SDS-PAGE and Western blotting. (D) Comigration with the presequence translocase. Mitochondria were solubilized in digitonin and loaded on top of a linear sucrose gradient. (E) The 18-kD J-protein is present in a functional translocase-preprotein complex. In the presence of methotrexate (MTX), purified b2(167)Δ-DHFR was arrested in mitochondria carrying Tom22His10. Mitochondria were lysed in digitonin and subjected to NiNTA–agarose chromatography. Following immunodecoration, samples were quantified using NIH-Image1.62f. The recovery of Tom40 was set to 100% (control).

Figure 2.

Figure 2.

The J-domain is exposed to the mitochondrial matrix. (A) The 35S-labeled precursor of the 18-kD J-protein of PAM was imported into isolated wild-type mitochondria for the indicated times. After proteinase K treatment, samples were analyzed by SDS-PAGE and digital autoradiography. Radiolabeled precursor (lane 8) and Western blot of the authentic, mature 18-kD protein (lane 9) are shown for comparison. (B) Mitochondria were sonicated in the presence of 500 mM NaCl or subjected to treatment at alkaline pH. Samples were left untreated (T) or subjected to centrifugation at 100,000 g. S, supernatant; P, pellet. (C) Part of the J-protein is exposed to the intermembrane space. Mitochondria were either directly treated with proteinase K or subjected to hypotonic swelling, sonication, or Triton X-100 lysis before proteinase K treatment. Samples were separated by SDS-PAGE and subjected to Western blotting. (D) The COOH-terminal portion with the J-domain is exposed to the matrix. Mitochondria were left untreated or subjected to hypotonic swelling before proteinase K treatment. Samples were subjected to SDS-PAGE and Western blotting with affinity-purified anti–18-kD J-domain antibodies.

Figure 3.

Figure 3.

The essential mitochondrial J-protein is selectively required for import of matrix proteins. (A) Accumulation of the precursors of mitochondrial proteins in J mutant cells (pam18–1; ts) shifted to 37°C for 16 h. (B) Protein levels. Isolated mitochondria (15-μg protein, odd-numbered lanes; 30-μg protein, even-numbered lanes) were analyzed by Western blotting. Nonrelevant gel lanes were excised digitally. (C) J mutant mitochondria (ts) generate a Δψ. The Δψ was assessed by fluorescence quenching using the dye DiSC3(5). (D) Impaired import of a matrix protein. 35S-labeled precursor was imported into mitochondria. Samples were treated with proteinase K and analyzed by digital autoradiography. (E) Import of a carrier protein is not affected. Dicarboxylate carrier (DIC) import and assembly into its dimeric form were analyzed by blue native PAGE and digital autoradiography. (F) Sorting of cytochrome b 2 at the inner membrane is not inhibited in J mutant mitochondria. Import of radiolabeled precursor was performed as described for D. (G) A relation of the J dependence on preprotein length. Radiolabeled precursors were imported as described above. (H) Import of b2(47)-DHFR is inhibited in ssc1–3 mitochondria. (I) J mutant mitochondria are impaired in import of saturating amounts of b2(47)-DHFR. Urea denaturated purified precursor was imported for the indicated times. Samples were treated with proteinase K and analyzed by Western blotting. The quantification shown is the average of at least three independent experiments. Import into wild-type mitochondria after the longest incubation time was set to 100% (control). p, precursor; i, intermediate; m, mature.

Figure 4.

Figure 4.

The J-domain stimulates the ATPase activity of mtHsp70 and influences the Tim44–mtHsp70 interaction. (A) ATPase activity of mtHsp70 under steady-state conditions using [α32P]ATP, purified mtHsp70, Mge1, and the J-domain of the 18-kD protein of PAM. Purified Tim44 did not stimulate the ATPase activity. (B) Disturbance of the Tim44–mtHsp70 interaction in J mutant (ts) mitochondria. Mitochondria were lysed under nondenaturing conditions and subjected to coprecipitation with antibodies directed against Tim44 or mtHsp70 (Voisine et al., 1999). Precipitates were analyzed by SDS-PAGE and immunodecoration (5% of the mitochondrial extracts are shown). Quantification: the amount of coprecipitated protein from wild type under ATP-depleted conditions was set to 100% (control). (C) Blue native PAGE of mitochondria lysed with digitonin, followed by immunodecoration with antibodies directed against Tim23, Tim22, and Tom40. (D) Protein A tagged Tim23 was expressed in wild-type and J mutant cells. Mitochondria were isolated, lysed with digitonin, and subjected to IgG chromatography.

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