A mutant form of mitochondrial GrpE suppresses the sorting defect caused by an alteration in the presequence of cytochrome b2.

Transport of preproteins across the inner mitochondrial membrane requires the action of the matrix heat shock protein Hsp70. Together with its co-chaperone mitochondrial GrpE (Mge1), mtHsp70 transiently binds to the inner membrane translocase subunit Tim44 in a nucleotide-regulated manner, forming an ATP-dependent import driving machinery. We report that a mutant form of Mge1 (Mge1-100) is completely absent in mtHsp70-Tim44 complexes, although its ability to interact with soluble mtHsp70 is only partially reduced. While this mge1-100 mutation only partially retards preprotein translocation into the matrix, it exerts a selective effect on sorting of cytochrome b2 to the intermembrane space. A cytochrome b2 with an altered sorting signal, which is only processed to the intermediate stage and mistargeted to the matrix of wild-type mitochondria, is processed to the mature form and correctly targeted to the intermembrane space of mge1-100 mitochondria. These results suggest that (1) Mge1-100 discriminates between soluble and membrane-bound mtHsp70 and (2) the membrane-bound mtHsp70-Mge1 driving system competes with the sorting machinery for translocation of preproteins like cytochrome b2.

[1]  B. Guiard.  Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)‐lactate cytochrome c oxidoreductase (cytochrome b2). , 1985, The EMBO journal.

[2]  E. Hurt,et al.  How proteins find mitochondria and intramitochondrial compartments , 1986 .

[3]  B. Guiard,et al.  One nuclear gene controls the removal of transient pre‐sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. , 1986, The EMBO journal.

[4]  F. Hartl,et al.  Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide , 1987, Cell.

[5]  N. Pfanner,et al.  Mitochondrial protein import. , 1989, Biochimica et biophysica acta.

[6]  A. Schneider,et al.  Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. , 1991, The EMBO journal.

[7]  F. Hartl,et al.  Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space , 1992, Cell.

[8]  R. Hallberg,et al.  Cytochromes c 1 and b 2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism , 1992, Cell.

[9]  E. Craig,et al.  Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix , 1993, The Journal of cell biology.

[10]  B. Glick,et al.  Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP , 1993, Protein science : a publication of the Protein Society.

[11]  E. Beasley,et al.  The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions. , 1993, The EMBO journal.

[12]  W. Neupert,et al.  Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal. , 1993, The EMBO journal.

[13]  M. Bauer,et al.  Mitochondrial Hsp70/MIM44 complex facilitates protein import , 1994, Nature.

[14]  D. Ang,et al.  Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes. , 1994, Molecular and cellular biology.

[15]  N. Pfanner,et al.  The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site , 1994, FEBS letters.

[16]  E. Craig,et al.  Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44 , 1994, The Journal of cell biology.

[17]  W. Oppliger,et al.  Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A. Gruhler,et al.  A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting. , 1995, The EMBO journal.

[19]  N. Pfanner,et al.  The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70. , 1995, The EMBO journal.

[20]  P. Dekker,et al.  Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM). , 1995, European journal of biochemistry.

[21]  E. Craig,et al.  Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins , 1995, Molecular and cellular biology.

[22]  B. Glick.  Can Hsp70 proteins act as force-generating motors? , 1995, Cell.

[23]  Walter Neupert,et al.  The MIM complex mediates preprotein translocationacross the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system , 1995, Cell.

[24]  N. Pfanner,et al.  [19] Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae , 1995 .

[25]  M. Meijer,et al.  Protein Sorting: Pulling in the proteins , 1995, Current Biology.

[26]  The Mitochondrial Protein Import Machinery , 1995, The Journal of Biological Chemistry.

[27]  W. Oppliger,et al.  The mitochondrial protein import motor: Dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis , 1996, Protein science : a publication of the Protein Society.

[28]  W. Neupert,et al.  The nucleotide exchange factor MGE exerts a key function in the ATP‐dependent cycle of mt‐Hsp70‐Tim44 interaction driving mitochondrial protein import. , 1996, The EMBO journal.

[29]  C. Georgopoulos,et al.  Purification and Biochemical Properties of Saccharomyces cerevisiae's Mge1p, the Mitochondrial Cochaperone of Ssc1p* , 1996, The Journal of Biological Chemistry.

[30]  B. Dobberstein,et al.  Common Principles of Protein Translocation Across Membranes , 1996, Science.

[31]  W. Neupert,et al.  Uniform nomenclature for the protein transport machinery of the mitochondrial membranes. , 1996, Trends in biochemical sciences.

[32]  W. Neupert,et al.  Mechanisms of protein import across the mitochondrial outer membrane. , 1996, Trends in cell biology.

[33]  N. Pfanner,et al.  Differential requirement for the mitochondrial Hsp70‐Tim44 complex in unfolding and translocation of preproteins. , 1996, The EMBO journal.

[34]  N. Pfanner,et al.  Mitochondrial biogenesis: The Tom and Tim machine , 1997, Current Biology.

[35]  A. Azem,et al.  Sequential action of two hsp70 complexes during protein import into mitochondria , 1997, The EMBO journal.

[36]  S. Barik.  Mutagenesis and gene fusion by megaprimer PCR. , 1997, Methods in molecular biology.

[37]  N. Pfanner,et al.  Role of mitochondrial GrpE and phosphate in the ATPase cycle of matrix Hsp70. , 1997, Journal of molecular biology.

[38]  E. Craig,et al.  Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae. , 1997, Journal of molecular biology.