Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co- chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.

[1]  K. Furtak,et al.  Folding in vivo of bacterial cytoplasmic proteins: Role of GroEL , 1993, Cell.

[2]  D. Vestweber,et al.  A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. , 1990, The EMBO journal.

[3]  G. Lorimer,et al.  Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. , 1991, Biochemistry.

[4]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[5]  T. Langer,et al.  The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding , 1993, Nature.

[6]  F. Hartl,et al.  Protein sorting to mitochondria: evolutionary conservations of folding and assembly. , 1990, Science.

[7]  G. Lorimer,et al.  Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent. , 1990, Biochemistry.

[8]  Meijia Yang,et al.  Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes. , 1988, The EMBO journal.

[9]  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.

[10]  W. Welch,et al.  The two mammalian mitochondrial stress proteins, grp 75 and hsp 58, transiently interact with newly synthesized mitochondrial proteins. , 1991, Cell regulation.

[11]  G. Lorimer,et al.  Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion. , 1993, Biochemistry.

[12]  F. Hartl,et al.  Identification of nucleotide-binding regions in the chaperonin proteins GroEL and GroES , 1993, Nature.

[13]  P. Tempst,et al.  Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis. , 1989, Analytical biochemistry.

[14]  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.

[15]  S. Busby,et al.  Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments. , 1982, Journal of Molecular Biology.

[16]  P. Høj,et al.  Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Lorimer,et al.  Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  T. Atkinson,et al.  Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding. , 1993, Biochemistry.

[19]  W. Fenton,et al.  GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase. , 1993, The Journal of biological chemistry.

[20]  F. Hartl,et al.  Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis , 1989, Nature.

[21]  G. Lorimer,et al.  Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP , 1989, Nature.

[22]  P. Viitanen,et al.  Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Schatz,et al.  The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting , 1986, Cell.

[24]  T. Junne,et al.  Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES , 1993, FEBS letters.

[25]  S. Chen,et al.  ATP induces large quaternary rearrangements in a cage-like chaperonin structure , 1993, Current Biology.

[26]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[27]  F. Hartl,et al.  Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites , 1989, The Journal of cell biology.

[28]  C. Ueguchi,et al.  Effects of mutations in heat‐shock genes groES and groEL on protein export in Escherichia coli. , 1989, The EMBO journal.

[29]  G. Schatz,et al.  Sequential action of mitochondrial chaperones in protein import into the matrix. , 1991, The EMBO journal.

[30]  E. Craig,et al.  Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae. , 1990, The Journal of biological chemistry.

[31]  F. Hartl,et al.  Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate , 1991, Nature.

[32]  F. Hartl,et al.  Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding , 1992, Nature.

[33]  A. Plückthun,et al.  The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta‐lactamase precursor. , 1990, The EMBO journal.

[34]  F. Hartl,et al.  Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase , 1986, Cell.

[35]  G. Howlett,et al.  Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog. , 1980, Analytical biochemistry.

[36]  P. Tempst,et al.  Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: Improved methods, applications and gene cloning strategies , 1990, Electrophoresis.

[37]  D. Botstein,et al.  A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. , 1987, Gene.

[38]  P. Høj,et al.  The complete primary structure of rat chaperonin 10 reveals a putative βαβ nucleotide-binding domain with homology to p21ras , 1993 .

[39]  A. Myers,et al.  Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor , 1989, Nature.

[40]  F. Hartl,et al.  Mitochondrial protein import. , 1987, Biochimica et biophysica acta.

[41]  L. Rosenberg,et al.  Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase. , 1992, The Journal of biological chemistry.

[42]  S. Gasser,et al.  Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. , 1982, The Journal of biological chemistry.

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

[44]  Y. Shu,et al.  Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins , 1993, Molecular and cellular biology.

[45]  G. Lorimer,et al.  Identification of a groES-like chaperonin in mitochondria that facilitates protein folding. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[46]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[47]  F. Hartl,et al.  Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria , 1989, Nature.

[48]  Elizabeth A. Craig,et al.  Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins , 1990, Nature.

[49]  W. Baumeister,et al.  Chaperonin‐mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. , 1992, The EMBO journal.

[50]  F. Hartl,et al.  Protein folding in the cell: the role of molecular chaperones Hsp70 and Hsp60. , 1992, Annual review of biophysics and biomolecular structure.

[51]  T. Langer,et al.  A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins , 1993, The Journal of cell biology.

[52]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[53]  A. Fersht,et al.  Cooperativity in ATP hydrolysis by GroEL is increased by GroES , 1991, FEBS letters.

[54]  F. Hartl,et al.  Prevention of protein denaturation under heat stress by the chaperonin Hsp60. , 1992, Science.

[55]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[56]  F. Hartl,et al.  Molecular chaperone functions of heat-shock proteins. , 1993, Annual review of biochemistry.

[57]  D. Kinney,et al.  Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. , 1986, Gene.

[58]  Lila M. Gierasch,et al.  Characterization of a functionally important mobile domain of GroES , 1993, Nature.

[59]  Roger W. Hendrix,et al.  Homologous plant and bacterial proteins chaperone oligomeric protein assembly , 1988, Nature.