title: The Hsp70 chaperone Ssq1p is dispensable for iron-sulfur cluster formation on the scaffold protein Isu1p.
abstract: The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.
authors.affiliation: Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland.
authors.forename: Rafal
authors.initials: R
authors.lastname: Dutkiewicz
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Marszalek
authors.forename: Brenda
authors.initials: B
authors.lastname: Schilke
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Craig
authors.forename: Roland
authors.initials: R
authors.lastname: Lill
authors.forename: Ulrich
authors.initials: U
authors.lastname: Mühlenhoff
mesh_terms: D020816:Amino Acid Motifs; D004305:Dose-Response Relationship, Drug; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000502:physiology*; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones / Q000737:chemistry / Q000502:physiology*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D013053:Spectrophotometry; D013440:Sulfides / Q000737:chemistry; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry; D013997:Time Factors
chemical_list: D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C121520:ISU1 protein, S cerevisiae; D007506:Iron-Sulfur Proteins; C115914:JAC1 protein, S cerevisiae; C086039:MGE1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; C115913:SSQ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013440:Sulfides; D007501:Iron; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C115912:NFS1 protein, S cerevisiae