Report |
Address correspondence to Nikolaus Pfanner, Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany. Tel.: 49-761-203-5224. Fax: 49-761-203-5261. email: Nikolaus.Pfanner{at}biochemie.uni-freiburg.de
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Abstract |
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Key Words: Hsp70; J-protein; mitochondria; protein translocation; Saccharomyces cerevisiae
Abbreviations used in this paper:
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Introduction |
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Three different subunits of the presequence translocaseassociated motor (PAM) of mitochondria are known, each of which is essential for cell viability: mtHsp70; the nucleotide exchange factor mitochondrial GrpE (Mge1); and the peripheral inner membrane protein Tim44, which serves as a binding partner for mtHsp70 (Jensen and Johnson, 1999; Matouschek et al., 2000; Pfanner and Geissler, 2001; Neupert and Brunner, 2002). Since Tim44 is associated with the Tim23Tim17 complex, mtHsp70 bound to Tim44 is located close to the exit of this inner membrane protein import channel. Chaperones of the Hsp70 class, including bacterial DnaK, Hsp70s of the eukaryotic cytosol, and BiP (Kar2) of the endoplasmic reticulum, typically cooperate with cochaperones of the J-class (DnaJ homology domain) (Bukau and Horwich, 1998; Pilon and Schekman, 1999; Matlack et al., 1999; Hartl and Hayer-Hartl, 2002). Mitochondria contain three J-proteins, termed Jac1, Mdj1, and Mdj2. However, these J-proteins are neither essential for cell viability nor involved in protein translocation across the inner membrane, but rather function in protein folding and maturation in the mitochondrial matrix (Rowley et al., 1994; Westermann and Neupert, 1997; Kim et al., 2001; Lutz et al., 2001; Voisine et al., 2001). It has thus been generally assumed that the essential translocase function of mtHsp70 does not require a J-protein, but that Tim44 acts as a specialized cochaperone at the protein import site. Therefore, all current models on the function of PAM are based on the assumption that only one membrane interaction site, Tim44, exists for mtHsp70 (Glick, 1995; Voisine et al., 1999; Matouschek et al., 2000; Neupert and Brunner, 2002; Liu et al., 2003).
We found a fourth mitochondrial J-protein that is an essential inner membrane protein associated with the presequence translocase. This J-protein of 18 kD stimulates the ATPase activity of mtHsp70 and is required for protein translocation into the matrix. Thus, mtHsp70 has two functional interaction sites located close to the protein import channel, Tim44 and the novel J-protein.
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Results and discussion |
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The in vitro synthesized 35S-labeled precursor of the 18-kD J-protein was incubated with isolated yeast mitochondria and transported to a protease-protected location only in the presence of a (Fig. 2 A). The imported protein had the same mobility on SDS-PAGE as the precursor (Fig. 2 A), indicating that the precursor is not proteolytically processed during import, consistent with the lack of an identifiable presequence in the primary structure (Fig. 1 B). After sonication of mitochondria, the J-protein remained associated with the membrane fraction (Fig. 2 B, lane 3). Since mitochondrial outer membrane proteins are imported independent of a
, these findings indicate that the protein is associated with the mitochondrial inner membrane. Treatment of membranes at pH 11.5 leads to an extraction of peripheral membrane proteins, whereas integral membrane proteins that are embedded in the lipid phase remain in the membrane sheets. The 18-kD J-protein was largely extracted from the membranes at pH 11.5, although it contains one predicted transmembrane segment, whereas the ADP/ATP carrier, which contains six transmembrane segments, remained in the pellet fraction (Fig. 2 B, lane 6). However, the Rieske Fe/S-protein of the bc1 complex, which spans the inner membrane with one transmembrane segment, showed the same behavior as the J-protein. A small fraction of both of these two proteins remained in the pellet fraction at pH 11.5 (Fig. 2 B, lane 6). At pH 10.8, nearly half of these proteins remained inextractable, whereas Tim44 was still completely extracted (Fig. 2 B, lane 9). The single transmembrane segment of the Fe/S-protein is not entirely embedded in the lipid phase of the inner membrane, but spans the membrane in association with other proteins (Lange and Hunte, 2002), suggesting that the single hydrophobic segment of the 18-kD J-protein similarly spans the membrane in association with other proteins.
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The essential J-protein is involved in protein import into mitochondria
To assess the role of the 18-kD J-protein in protein transport, we generated conditional alleles by error-prone PCR and selected a mutant strain with a temperature-sensitive lethal growth phenotype at 37°C. When the mutant cells were shifted to the nonpermissive temperature, mitochondrial precursor proteins accumulated in vivo (Fig. 3 A). Mutant cells grown at 24°C contained wild-type amounts of all mitochondrial marker proteins analyzed (Fig. 3 B). To minimize indirect effects on mitochondrial functions, the mutant cells were grown at the permissive temperature of 24°C, and isolated mitochondria were preincubated at 37°C before import reactions. J mutant mitochondria were as competent in the generation of a as wild-type mitochondria (Fig. 3 C). The matrix-targeted preprotein b2(167)
-DHFR was synthesized and radiolabeled in rabbit reticulocyte lysate and incubated with isolated mitochondria. Its import, determined by proteolytic processing and transport to a protease-protected location, was up to threefold reduced in the mutant mitochondria compared with wild-type (Fig. 3 D). Import of a carrier protein that uses the second inner membrane translocase (TIM22 complex) was not affected (Fig. 3 E), indicating a specific impairment of the presequence pathway in the mutant mitochondria. When the inner membrane sorting signal of cytochrome b2 is included in the preprotein, the resulting protein b2(167)-DHFR becomes arrested in the inner membrane by this hydrophobic sorting signal and can be imported in the absence of functional mtHsp70 (Voos et al., 1993). Indeed, the import of b2(167)-DHFR into the mutant mitochondria was similar to that in wild-type mitochondria (Fig. 3 F), raising the possibility that the import defect is related to a functional impairment of mtHsp70.
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The essential J-protein stimulates mtHsp70
We expressed and purified the J-domain and analyzed its effect on the ATPase activity of purified mtHsp70 under steady-state conditions. An incubation of the J-domain with mtHsp70 only slightly stimulated the ATP turnover, similar to an incubation of purified Mge1 with mtHsp70 (Fig. 4 A) (Weiss et al., 2002). However, addition of the J-domain to mtHsp70 in the presence of Mge1 led to an 10-fold stimulation of the ATPase activity (Fig. 4 A). Tim44 did not stimulate the ATPase activity of mtHsp70 (data not shown). We conclude that the J-domain stimulates the activity of mtHsp70 and thus the novel 18-kD J-protein is a subunit of PAM.
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In summary, we report that an 18-kD integral inner membrane protein with a J-domain constitutes an essential component of the mitochondrial import motor PAM. The J-protein is associated with the presequence translocase. Its inactivation impairs the interaction of mtHsp70 with Tim44 and inhibits the translocation of preproteins into the mitochondrial matrix. The identification of this J-protein indicates that the reaction cycle of mtHsp70 at the protein import site of the mitochondrial inner membrane requires a functional interaction with two essential membrane proteins, the 18-kD J-protein and Tim44. The J-protein promotes the reaction cycle of mtHsp70 by stimulating its ATPase activity.
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Materials and methods |
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In vitro import into yeast mitochondria
Growth of yeast cells and isolation of mitochondria were performed according to Ryan et al. (2001). For isolation of mutant and wild-type mitochondria, yeast cells were grown at 24°C. Synthesis and transport of 35S-labeled proteins into isolated mitochondria was performed as described (Ryan et al., 2001). For import into mutant mitochondria, mitochondria were preincubated at 37°C for 15 min, and import reactions were performed at 25°C. Import of saturating amounts of urea-denaturated preprotein was performed using 280 pmol of purified b2(47)-DHFR preprotein/mg mitochondrial protein (Dekker et al., 1997). Samples were subsequently separated by SDS-PAGE and either subjected to digital autoradiography (radiolabeled preproteins) or probed with anti-DHFR IgGs (saturating amounts of preprotein).
Fractionation and coimmunoprecipitation
Hypotonic swelling of mitochondria, subsequent treatment with proteinase K and carbonate extraction were performed as described (Ryan et al., 2001). Mitochondria were sonicated on ice (3 x 30 s with 40% duty cycle in a Branson Sonifier 250) in the presence of 5 µg/ml proteinase K in 10 mM Tris, pH 7.4, 500 mM NaCl. Coimmunoprecipitations were performed as described (Voisine et al., 1999). Purification of recombinant proteins and immunization of rabbits was performed as described in Geissler et al. (2002) or by the manufacturer (Amersham Biosciences).
Miscellaneous
Purification of the TIM23 complex was performed according to Geissler et al. (2002) with the exception that elution of the IgGSepharose bound complex was achieved by TEVprotease cleavage overnight at 4°C. Isolation of a TOMTIM translocation intermediate, complex analysis on sucrose gradients, measurements, digest of proteins in gel, preparation for nano-HPLC and ESI mass spectrometry were performed as described in Geissler et al. (2002). Blue native PAGE was performed as described by Dekker et al. (1997). For ATPase assays, all proteins were purified to homogeneity. MtHsp70 (Ssc1) was purified from mitochondria by NiNTA chromatography. Mge1 was isolated according to Dekker and Pfanner (1997) and the J-domain (GST-Pam1884168) was purified as described above. ATPase activity was determined according to Dekker and Pfanner (1997).
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Acknowledgments |
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This work was supported by the Sonderforschungsbereich 388, Max Planck Research Award, Nationales Genomforschungsnetz, BMBF, and the Fonds der Chemischen Industrie. M. Lind is a recipient of a postdoctoral fellowship from the Wenner-Gren foundations.
Submitted: 1 August 2003
Accepted: 6 October 2003
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