The Presequence Translocase-associated Protein Import Motor of Mitochondria

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 His-Pro-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. 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 His-Pro-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 (1Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (978) Google Scholar, 2Koehler C.M. Merchant S. Schatz G. Trends Biochem. Sci. 1999; 24: 428-432Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Scheffler I.E. Mitochondrion. 2000; 1: 3-31Crossref Scopus (205) Google Scholar, 4Jensen R. Dunn C. Biochim. Biophys. Acta. 2002; 1592: 25-34Crossref PubMed Scopus (95) Google Scholar, 5Endo T. Yamamoto H. Esaki M. J. Cell Sci. 2003; 116: 3259-3267Crossref PubMed Scopus (149) Google Scholar, 6Truscott K.N. Brandner K. Pfanner N. Curr. Biol. 2003; 13: R326-R337Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). 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). 1The abbreviations used are: TIM, translocase of inner mitochondrial membrane; Pam16S, soluble domain of Pam16 lacking the 25 amino-terminal amino acid residues; Pam18J, J-domain of Pam18; DHFR, dihydrofolate reductase; Mdj1, mitochondrial DnaJ protein 1; Mge1, mitochondrial GrpE; mtHsp70, mitochondrial heat shock protein 70 (Ssc1); PAM, presequence translocase-associated motor; YP, yeast-rich medium; Ni-NTA, nickel-nitrilotriacetic acid. The presequence translocase forms a voltage-activated translocation channel across the inner membrane through which the loosely folded precursor polypeptides pass (7Truscott K.N. Kovermann P. Geissler A. Merlin A. Meijer M. Driessen A.J.M. Rassow J. Pfanner N. Wagner R. Nat. Struct. Biol. 2001; 8: 1074-1082Crossref PubMed Scopus (254) Google Scholar). 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) (8Strub A. Lim J.H. Pfanner N. Voos W. Biol. Chem. 2000; 381: 943-949Crossref PubMed Scopus (43) Google Scholar, 9Matouschek A. Pfanner N. Voos W. EMBO Rep. 2000; 1: 404-410Crossref PubMed Scopus (129) Google Scholar, 10Rehling P. Brandner K. Pfanner N. Nat. Rev. Mol. Cell Biol. 2004; 5: 519-530Crossref PubMed Scopus (281) Google Scholar, 11Wiedemann N. Frazier A.E. Pfanner N. J. Biol. Chem. 2004; 279: 14473-14476Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). 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 (12Voos W. Martin H. Krimmer T. Pfanner N. Biochim. Biophys. Acta. 1999; 1422: 235-254Crossref PubMed Scopus (129) Google Scholar, 13Herrmann J.M. Neupert W. Biochim. Biophys. Acta. 2000; 1459: 331-338Crossref PubMed Scopus (19) Google Scholar). Recent studies reveal that the PAM complex is one of the most complex Hsp70 systems (14Mayer M.P. Nat. Struct. Mol. Biol. 2004; 11: 6-8Crossref PubMed Scopus (16) Google Scholar). 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 (15Neupert W. Brunner M. Nat. Rev. Mol. Cell Biol. 2002; 3: 555-565Crossref PubMed Scopus (300) Google Scholar, 16Voisine C. Craig E.A. Zufall N. von Ahsen O. Pfanner N. Voos W. Cell. 1999; 97: 565-574Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 17Liu Q. D'Silva P. Walter W. Marszalek J. Craig E.A. Science. 2003; 300: 139-141Crossref PubMed Scopus (145) Google Scholar). The mitochondrial GrpE (Mge1) promotes the release of nucleotides from mtHsp70 and, thus, allows the completion of the mtHsp70 reaction cycle (18Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (82) Google Scholar, 19Dekker P.J.T. Pfanner N. J. Mol. Biol. 1997; 270: 321-327Crossref PubMed Scopus (44) Google Scholar, 20Schneider H.C. Westermann B. Neupert W. Brunner M. EMBO J. 1996; 15: 5796-5803Crossref PubMed Scopus (99) Google Scholar). Most Hsp70s cooperate with cochaperones of the DnaJ protein family, so called J-proteins, which stimulate the ATPase activity of Hsp70 (21Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (400) Google Scholar, 22Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2429) Google Scholar, 23Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (868) Google Scholar, 24Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2792) Google Scholar). Three mitochondrial J-proteins have been known for several years, Mdj1, Mdj2, and Jac1 (25Rowley N. Prip-Buus C. Westermann B. Brown C. Schwarz E. Barrell B. Neupert W. Cell. 1994; 77: 249-259Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 26Westermann B. Neupert W. J. Mol. Biol. 1997; 272: 477-483Crossref PubMed Scopus (41) Google Scholar, 27Kim R. Saxena S. Gordon D.M. Pain D. Dancis A. J. Biol. Chem. 2001; 276: 17524-17532Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, none of them plays a role in driving preprotein translocation via the PAM machinery. Recently, an inner membrane-bound J-protein termed Pam18 (Tim14) was identified as a component of the PAM complex that plays an essential role in preprotein translocation (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar, D. M. Neupert W. K. EMBO J. 2003; PubMed Scopus Google Scholar). a Pam16 was and to an essential of the PAM complex A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). A of Pam18 and Pam16 in cochaperones for protein translocation into the mitochondrial matrix but not for protein into the inner membrane (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar, D. M. Neupert W. K. EMBO J. 2003; PubMed Scopus Google Scholar, A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). Pam18 and Pam16 a that with the presequence translocase and the other motor A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). However, Pam18 a J-domain that is for the interaction with Hsp70 and is in proteins of the J-protein K. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. D. C. K. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Pam16 a domain The Pam16 domain a and as the Pam18 but the motif HPD that is in known J-proteins is in Pam16. for a protein of the DnaJ family, the J-domain of Pam18 the ATPase activity of mtHsp70 (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar). studies with Pam16 not a stimulatory effect the ATPase activity of mtHsp70 A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). the function of Pam16 in the protein import motor report that Pam16 inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70. Pam16 to a cochaperone by inclusion of the HPD The Pam16 in vitro and in vivo but replace the function of Pam18. thus, does not a typical Hsp70 cochaperone of the J-protein but, rather, to the activity of Pam18. and used in are in the is the of a a of of the was in the by a the A. A. A. P. 1998; PubMed Scopus Google Scholar). by The with the with the The yeast-rich and The cell was in different and to the The for and used in P. PubMed Google P. PubMed Google K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google in a new the and the of by of the the wild-type from a by acid for the The by the with the and of Pam16S, the of to amino acid was by the and the the was by in with the and which the HPD The by a and the was the soluble of and with and and into the (7Truscott K.N. Kovermann P. Geissler A. Merlin A. Meijer M. Driessen A.J.M. Rassow J. Pfanner N. Wagner R. Nat. Struct. Biol. 2001; 8: 1074-1082Crossref PubMed Scopus (254) Google to an amino-terminal After of protein in the proteins by and the interaction Pam16 and Pam18 was as A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). from the mtHsp70 with a from Mitochondria in and After for and of with the was with in for proteins by with and proteins with and the was and in a and and as P.J.T. Pfanner N. J. Mol. Biol. 1997; 270: 321-327Crossref PubMed Scopus (44) Google Scholar). the proteins in E. After of the the proteins by ATPase Ssc1 and to with other cochaperones as in of A and and by in acid and by and preproteins by in vitro and into as Voos W. Pfanner N. Cell Biol. 2001; PubMed Google Scholar). a heat for to was by the of and with K. in and proteins by as C. Craig E.A. Zufall N. von Ahsen O. Pfanner N. Voos W. Cell. 1999; 97: 565-574Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). by a of preproteins with to the of a dihydrofolate domain A. Rassow J. Pfanner N. Voos W. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). for interaction of the with mtHsp70 as that not with K. was in wild-type and from the The of TIM23 translocase via a protein was as A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). Pam16 the Pam18-mediated of the ATPase of Pam16 not stimulate the ATPase activity of mtHsp70 in a soluble A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). Pam16 a in the amino-terminal the domain is used for of Pam16 with the inner membrane A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). was that the with the function of Pam16 in the for the ATPase activity of mtHsp70. thus, a of Pam16 lacking the amino-terminal 25 amino acid but the the typical for DnaJ proteins in E. with an amino-terminal the of Pam16 was fully soluble and termed The ATPase activity of mtHsp70 was by the of not stimulate the ATPase activity of mtHsp70 the nucleotide was We and Pam16 in a but a stimulation of the ATPase activity was in to the stimulation of the ATPase activity of mtHsp70 by the J-domain of Pam18, termed (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar). We the activity of by both proteins the stimulation of the mtHsp70 ATPase by of the ATPase stimulation by was a of a Pam16 the stimulatory effect of The effect of Pam16 the stimulation of the ATPase activity of mtHsp70 was not in the of proteins not that Pam16 in an manner to Pam18. for the of Pam16 Pam16 displays a similarity to the J-domain of Pam18 and other J-proteins A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google to the of mtHsp70 and, thus, for the of Pam18. Pam16 specifically antagonizes the function of Pam18 but not of other the stimulation of the ATPase activity of mtHsp70 by the mitochondrial matrix J-protein (25Rowley N. Prip-Buus C. Westermann B. Brown C. Schwarz E. Barrell B. Neupert W. Cell. 1994; 77: 249-259Abstract Full Text PDF PubMed Scopus (200) Google Scholar). the ATPase activity of mtHsp70 in a manner with the stimulation by (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar). However, the of not the stimulation of the ATPase activity does not the interaction of mtHsp70 for J-proteins but the activity of Pam18. of the HPD in Pam16 with and of HPD motif in the of a J-domain K. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google that is for the interaction with Hsp70 and the stimulation of the ATPase activity is in Pam16. We the of an HPD motif Pam16 to a protein with an activity to other proteins of the DnaJ family, a stimulatory effect the ATPase activity of mtHsp70. the and are Pam16 and Pam18 A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google the amino of Pam16 by HPD the to the HPD motif of Pam18 with an amino-terminal was in E. and to We was able to the protein complex with Pam18. was a and with the J-domain of Pam18 protein with (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar). to was only in the with We that Pam16 with an HPD motif is able to with the J-domain of Pam18. a function of Pam16 with an HPD the ATPase activity of mtHsp70. However, was not able to stimulate the ATPase activity of mtHsp70 the that an a activity as Pam16S, an of the Pam18-mediated stimulation of mtHsp70. was in the mtHsp70 ATPase with Pam18J, the stimulation of the mtHsp70 ATPase activity by was in a manner with the effect of is in vitro Pam16-HPD in but for in vivo Pam16 with an HPD motif was Pam16-HPD in The of the wild-type of Pam16 was by the the of a the of in the the of the was The of wild-type Pam16 from a the as of Pam16-HPD fully cell the of wild-type Pam16 that Pam16-HPD is in the of the wild-type protein are and cell Pam16-HPD is Pam16-HPD fully for wild-type a lacking the and by of Pam16 from a the Pam16 was by a The the as a wild-type Pam16 that Pam16-HPD fully the function of Pam16 in We Pam16-HPD was able to replace Pam18 in We a lacking the and Pam18 from a the Pam18 was by a Pam16 a however, the not Pam16 the Pam16 the HPD motif for Pam18 in Pam16 the of mtHsp70 via a role for Pam16 during the import that is different from the Hsp70 stimulatory function of other the of a of Pam16 the interaction of mtHsp70 with precursor proteins during the import We the preprotein a protein the presequence of the and dihydrofolate into wild-type and and the of protein that is to mtHsp70. After completion of the translocation reaction into the and a was with mtHsp70. in wild-type only a of proteins to mtHsp70, a of to mtHsp70 preprotein into the protein with mtHsp70 in the matrix the preprotein a protein an amino-terminal portion of and DHFR, a translocation in a complex the preprotein and the of the outer and inner mitochondrial membranes A. Rassow J. Pfanner N. Voos W. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, M. Schatz G. PubMed Scopus Google Scholar, D.M. C. Neupert W. PubMed Scopus Google Scholar, A. Rehling P. Guiard B. Frazier A.E. A. Pfanner N. Voos W. Meisinger C. EMBO J. 2003; PubMed Scopus Google Scholar). We used to the activity of mtHsp70 to preproteins in of the translocation is in a with mtHsp70 in wild-type C. Craig E.A. Zufall N. von Ahsen O. Pfanner N. Voos W. Cell. 1999; 97: 565-574Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, C. Neupert W. D.M. Science. 1994; PubMed Scopus Google Scholar). a of that the of protein in was in wild-type The of Pam16 the interaction mtHsp70 and translocation different from effect the of mtHsp70 to fully We the of mtHsp70 preproteins by the interaction of Pam16 with Pam18. studies that in a different of Pam16 the interaction of Pam18 with the TIM23 complex was A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). the used also in the the TIM23 translocase complex from wild-type and and both for the of Pam18. in wild-type a interaction of Pam18 with the TIM23 complex was as the of Pam18 was not in with the TIM23 complex in in the of that Pam18 stably with the translocase complex in the of the protein We that Pam16 as an protein the activity of mtHsp70 the import via the of Pam18. The identified protein Pam16 is associated with the TIM23 translocase complex that the channel for the of preproteins into the mitochondrial matrix A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). Pam16 a protein domain that a to Hsp70 cochaperones of the DnaJ However, an motif of the the tripeptide is in Pam16 A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). We the Pam16 as a J-protein in the activity of mtHsp70. that the of the domain Pam16 does not function as a stimulate the ATPase activity of mtHsp70 of the mitochondrial matrix. effect is not to the of the HPD motif for the activity of proteins the inclusion of the motif into the Pam16 not in a stimulatory function mtHsp70 ATPase activity and, not Pam16 to a Pam16-HPD activity and for the essential function of wild-type Pam16 in cell these that Pam16 represents a type of the of which role Pam16 plays in the preprotein translocation activity of mtHsp70. Pam16 been to with Pam18, essential J-protein and component of the presequence translocase-associated motor A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). The membrane protein Pam18 the ATPase activity of mtHsp70 and is for the import of mitochondrial proteins (28Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Müller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar, 29D'Silva P. Schilke B. Walter W. Andrew A. Craig E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13839-13844Crossref PubMed Scopus Google Scholar, D. M. Neupert W. K. EMBO J. 2003; PubMed Scopus Google Scholar). Pam16 the function of Pam18 in mtHsp70. effect was for Pam18 and not for other J-proteins with mtHsp70, that Pam16 not the of mtHsp70. The effect of Pam16 is by the of a with Pam18 A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). Pam16 is to protein translocation in vivo and into A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar, C. D. M. Neupert W. K. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google effect Pam18 does not to an of mitochondrial preprotein translocation but is for a function of the import motor precursor proteins that fully into the matrix with mtHsp70 in wild-type mitochondria. most an stimulatory of the Pam18 the ATPase activity of mtHsp70, in a to However, the of mtHsp70 not to in the translocation reaction a import A.E. Dudek J. Guiard B. Voos W. Li Y. Lind M. Meisinger C. Geissler A. Sickmann A. Meyer H.E. M.G. K.N. Pfanner N. Rehling P. Nat. Struct. Mol. Biol. 2004; 11: PubMed Scopus Google Scholar). The translocation activity of is by the of mtHsp70 to preproteins as translocation in the mitochondrial to the of Pam18 that is in with the TIM23 translocase complex in Pam18 is not able to stimulatory function mtHsp70 the translocation A activity of Pam18 and mtHsp70 a of of the presequence translocase and the motor during the translocation reaction and to an of mitochondria. We that Pam16 is in the activity of Pam18 the reaction cycle of mtHsp70 a the activity of mtHsp70 is an and of the Pam18-mediated stimulation of mtHsp70 activity is a major of an translocation We J. K. N. and A. Frazier for and and N. Zufall for