Identification of Essential Residues in the Type II Hsp40 Sis1 That Function in Polypeptide Binding
2002; Elsevier BV; Volume: 277; Issue: 24 Linguagem: Inglês
10.1074/jbc.m111075200
ISSN1083-351X
AutoresSoojin Lee, Chun Yang Fan, J. Michael Younger, Hongyu Ren, Douglas Cyr,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoSis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins. Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins. carboxyl-terminal domain dithiothreitol enzyme-linked immunosorbent assay phosphate-buffered saline bovine serum albumin α-lactalbumin reduced lactalbumin denatured luciferase graphicalrepresentation and analysis ofstructural properties Hsp40s represent a structurally diverse family of co-chaperones that function with Hsp70 to facilitate cellular processes that include protein folding, the suppression of protein aggregation, endocytosis, protein translocation across membranes, signal transduction, DNA replication, protein degradation, and prion propagation (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 2Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar, 3Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3130) Google Scholar, 4Sondheimer N. Lopez N. Craig E.A. Lindquist S. EMBO J. 2001; 20: 2435-2442Crossref PubMed Scopus (169) Google Scholar). Hsp70 facilitates these processes by utilizing energy derived from ATP hydrolysis to bind and release regions of proteins that exhibit aspects of non-native structure (5Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar, 6Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 7Mayer M.P. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (311) Google Scholar). Hsp40s function by regulating the Hsp70 ATP hydrolytic cycle (8Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar, 9Cyr D.M., Lu, X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar) and by acting as molecular chaperones that bind and target non-native proteins to the peptide-binding site of Hsp70 (10Wickner S. Hoskins J. McKenney K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7903-7907Crossref PubMed Scopus (138) Google Scholar, 11Langer T., Lu, C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar). To regulate Hsp70 ATPase activity Hsp40 proteins utilize a conserved region, which was identified in Escherichia coli DnaJ and is termed the J-domain (12Georgopoulos C.P. Lundquist-Heil A. Yochem J. Feiss M. Mol. Gen. Genet. 1980; 178: 583-588Crossref PubMed Scopus (52) Google Scholar, 13Zylicz M. Yamamoto T. McKittrick N. Sell S. Georgopoulos C. J. Biol. Chem. 1985; 260: 7591-7598Abstract Full Text PDF PubMed Google Scholar). The J-domain, found in all Hsp40s, is around 70 amino acids in length and contains a conserved HPD tripeptide that is the signature motif of this protein family (14Cyr D.M. Gething M.-J. Guidebook to Molecular Chaperones and Protein Folding Factors. Sambrook & Tooze at Oxford University Press, Oxford1997: 89-95Google Scholar). The NMR structure of the J-domain shows it to contain four α-helical regions with the HPD motif being located in a loop that connects Helix II and Helix III (15Szyperski T. Pellecchia M. Wall D. Georgopoulos C. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11343-11347Crossref PubMed Scopus (141) Google Scholar, 16Hill R.B. Flanagan J.M. Prestegard J.H. Biochemistry. 1995; 34: 5587-5596Crossref PubMed Scopus (69) Google Scholar, 17Qian Y.Q. Patel D. Hartl F.U. McColl D.J. J. Mol. Biol. 1996; 260: 224-235Crossref PubMed Scopus (139) Google Scholar). How the J-domain regulates Hsp70 ATPase activity is not entirely clear, but a surface formed by helix II and the HPD motif is proposed to bind a cleft at the base of the Hsp70 ATPase domain and thereby stimulates ATP hydrolysis (18Greene M.K. Maskos K. Landry S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6108-6113Crossref PubMed Scopus (248) Google Scholar, 19Suh W.C. Burkholder W.F., Lu, C.Z. Zhao X. Gottesman M.E. Gross C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15223-15228Crossref PubMed Scopus (228) Google Scholar, 20Gassler C.S. Buchberger A. Laufen T. Mayer M.P. Schroder H. Valencia A. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15229-15234Crossref PubMed Scopus (151) Google Scholar). Energy derived from ATP hydrolysis then drives a conformational change in Hsp70 that is proposed to involve the closure of a lid structure that covers the peptide-binding groove and stabilizes Hsp70-peptide complexes (6Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 21Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar). How Hsp40s function as molecular chaperones to bind and deliver non-native proteins to Hsp70 is not well established (23Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Crossref PubMed Scopus (225) Google Scholar). The study of the mechanism for Hsp40 chaperone function is complicated by the fact that Type I, II, and III Hsp40s are not functionally equivalent (24Caplan A.J. Douglas M.G. J. Cell Biol. 1991; 114: 609-621Crossref PubMed Scopus (213) Google Scholar, 25Luke M.M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (147) Google Scholar, 26Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (97) Google Scholar, 27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 28Gall, W. E., Higginbotham, M. A., Chen, C. Y., Ingram, M. F., Cyr, D. M., Graham, T. R., Curr. Biol., 10, 1349, 1349, 1358.Google Scholar). Biochemical studies with purified Type I Hsp40s such asE. coli DnaJ, human Hdj-2, and yeast Ydj-1 demonstrate that these proteins function as chaperones independent of Hsp70 to suppress protein aggregation (11Langer T., Lu, C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 29Cyr D.M. FEBS Lett. 1995; 359: 129-132Crossref PubMed Scopus (115) Google Scholar). On the other hand, Type II Hsp40s such as human Hdj-1 and yeast Sis1 appear to be less efficient as chaperones and need to act with Hsp70 to suppress protein aggregation (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 30Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Type III Hsp40s do not appear capable of suppressing protein aggregation or facilitating protein folding and, therefore, may not function as molecular chaperones (2Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar). Differences in the structures of Type I, II, and III Hsp40s appear to account for the differences in chaperone activity exhibited by these co-chaperone proteins. Type I Hsp40s are modeled after DnaJ and contain a J-domain, a Gly and Phe (G/F)-rich region, a zinc finger-like domain, and a conserved carboxyl-terminal domain (CTD).1 Biochemical and genetic studies suggest that Type I Hsp40s utilize the zinc finger-like region and portions of CTD to bind non-native proteins (31Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (276) Google Scholar, 32Banecki B. Liberek K. Wall D. Wawrzynow A. Georgopoulos C. Bertoli E. Tanfani F. Zylicz M. J. Biol. Chem. 1996; 271: 14840-14848Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 33Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Type II Hsp40s contain the J-domain, G/F-rich region, and the CTD but lack the zinc finger-like region, which is replaced in part by a Gly and Met (G/M)-rich region (2Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar). Biochemical and genetic studies suggest that the G/F region and portions of the conserved carboxyl terminus enable Type II Hsp40s to function as chaperones (4Sondheimer N. Lopez N. Craig E.A. Lindquist S. EMBO J. 2001; 20: 2435-2442Crossref PubMed Scopus (169) Google Scholar, 27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 34Yan W. Craig E.A. Mol. Cell. Biol. 1999; 19: 7751-7758Crossref PubMed Scopus (123) Google Scholar). Type III Hsp40s contain the J-domain and other specialized structures that enable them to bind specific proteins, nucleic acids, and insert into intracellular membranes (2Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar). Thus, Hsp40s have evolved to contain different types of polypeptide-binding domains, and this structural divergence enables them to direct Hsp70 to bind a broad range of substrates. To investigate the mechanism for the chaperone function of Type II Hsp40s, we utilized the yeast Sis1 protein as a model protein (25Luke M.M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (147) Google Scholar). Sis1 is an essential 352-amino acid residue protein that functions in the cytosol with members of the Hsp70 Ssa family (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 35Horton L.E. James P. Craig E.A. Hensold J.O. J. Biol. Chem. 2001; 276: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Biochemical studies show that the polypeptide binding activity of Sis1 is retained by a fragment of the protein that contains residues 171–352 (Sis1-(171–352)) (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Consistent with these data, genetic studies have demonstrated that the CTD of Sis1 carries out functions that are essential to support cell viability (36Johnson J.L. Craig E.A. J. Cell Biol. 2001; 152: 851-856Crossref PubMed Scopus (85) Google Scholar). However, the mechanism by which Sis1 binds and delivers non-native polypeptides to Hsp70 is not clear. Insight into the nature of the Sis1 peptide-binding site was provided by the crystal structure of Sis1-(171–352), which reveals that CTD of Sis1 forms a crystallographic homodimer that has a wishbone-like structure (37Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (145) Google Scholar). Sis1-(171–352) monomers have an elongated shape and contain two barrel-like domains, CTDI and CTDII, and a C-terminal dimerization motif that correspond to residues 180–255, 260–329, and 330–352, respectively (37Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (145) Google Scholar). Deletion of the dimerization domain of Sis1 reduces its ability to help Hsp70 refold luciferase (37Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (145) Google Scholar), but monomeric Sis1 can still support the growth of yeast (36Johnson J.L. Craig E.A. J. Cell Biol. 2001; 152: 851-856Crossref PubMed Scopus (85) Google Scholar). Thus, Sis1 can carry out its essential functions as a monomer, and contrary to a previous suggestion (23Rudiger S. Schneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Crossref PubMed Scopus (225) Google Scholar), the dimerization domain is not likely to play a direct role in polypeptide binding. To bind non-native polypeptides, chaperone proteins typically utilize regions enriched in solvent-exposed hydrophobic amino acid side chains (38Saibil H.R. Curr. Opin. Struct. Biol. 2000; 10: 251-258Crossref PubMed Scopus (104) Google Scholar). Analysis of the Sis1-(171–352) structure revealed the existence of a hydrophobic patch of amino acids located on the surface of domain I, which was predicted to participate in Sis1 chaperone function (37Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (145) Google Scholar). To test this model, we carried out a mutational analysis of residues present in the hydrophobic patch in CTDI of Sis1. The results reported herein demonstrate that highly conserved residues within CTDI are essential for cell viability and are required for Sis1 to bind non-native polypeptides. To produce a vector to drive the overexpression of Sis1 in E. coli, the coding sequence of Sis1 was amplified from yeast genomic DNA by polymerase chain reaction (PCR) with the 5′-primer, SIS-N (5′-ACAGAACTAACCATGGTCAAGGAGACAAACT T-3′), and the 3′-prime primer, SIS-C (5′-TGCTTAGGATCCCTATTAAAAATTTTCATCTAT AGC-3′). This PCR product was then cloned into the NdeI and BamHI sites present in the polylinker of the E. coli expression vector pET9a (39Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar) to generate pET9aSis1. To express Sis1 from a plasmid in yeast under the control of its own promoter the primers SIS-UN (5′-ATGACCATCGATCATCCATCTGTTGTCCTGTGAAAAGA-3′) and SIS-C were utilized to generate a PCR fragment that contained bases that were –772 to 1056 from the Sis1 start codon (25Luke M.M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (147) Google Scholar). This PCR fragment contains both the Sis1 promoter and open reading frame and was subcloned into the SpeI and BamHI sites present in the polylinkers of the centromeric yeast expression plasmids pRS314 and pRS315 (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to generate pRS314Sis1 and pRS315Sis1. To construct the Sis1 point mutants characterized in this study (see Fig. 2), a 4-primer PCR-based mutagenic protocol was utilized (41Caplan A.J. Cyr D.M. Douglas M.G. Cell. 1992; 71: 1143-1155Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Briefly, the primers, SIS1-N and SIS1-C were employed in combination with a set of internally overlapping mutagenic primers to generate PCR products that contained a single point mutation in Sis1. The mutated Sis1 PCR products were then digested withStuI and BamHI to generate a DNA fragment that contained bases 148–1056 of Sis1. pET9aSIS1, pRS314Sis1, and pRS315SIS1 were then digested with StuI and BamHI, and the mutated and digested Sis1 PCR fragments were utilized to replace the region of the wild-typeSis1 open reading frame present in these plasmids that corresponded to bases 148 to 1056. Yeast Hsp70 Ssa1 was purified from yeast strain MW141 (42Werner-Washburne M. Stone D.E. Craig E.A. Mol. Cell. Biol. 1987; 7: 2568-2577Crossref PubMed Scopus (294) Google Scholar) grown in YP medium containing 2% galactose to an A600 of 3. Hsp70 Ssa1 was then purified using ATP-agarose, ion exchange, and hydroxyapatite chromatography as described previously (9Cyr D.M., Lu, X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Wild-type and mutant Sis1 were overexpressed in E. coli BL21(DE3)pLys by induction with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside followed by growth for 3 h at 30 °C. Purification of Sis1 was then carried out by ion exchange and hydroxyapatite chromatography as described previously (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Purified proteins were stored on ice or at –80 °C prior to use. The ability of Sis1 to cooperate with Hsp70 Ssa1 to facilitate the refolding of chemically denatured luciferase was monitored as described previously (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The ability of Sis1 to stimulate the ATPase activity of Hsp70 Ssa1 was monitored by thin layer chromatography with polyethyleneimine-cellulose plates as previously described (9Cyr D.M., Lu, X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Purified Sis1 (0.3 mg/ml) was incubated at 30 °C for 1 h. in 30 ml of buffer (10 mm Hepes, pH 7.4, 150 mm KCl, and 5 mm DTT) that was supplemented with proteinase K (0.01–1.0 mg/ml). Digestions were terminated by the addition of 0.5 mm phenylmethylsulfonyl fluoride, and samples were immediately added to SDS-PAGE sample buffer and run out on 12.5% SDS-PAGE. Previous studies have demonstrated that the proteolytic products liberated from Sis1 to be a 21-kDa band that corresponds to residues 171–352 and a pair of 7–9-kDa bands that represent fragments containing the J-domain (27Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). To compare the peptide binding activity of Sis1 and the Sis1 mutants, a binding assay representing a modified version of the enzyme-linked immunosorbent assay (ELISA) method for detecting complex formation between DnaJ and its substrates was established (43Wawrzynow A. Banecki B. Wall D. Liberek K. Georgopoulos C. Zylicz M. J. Biol. Chem. 1995; 270: 19307-19311Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The assay is based on the ability of purified Sis1 to bind non-native proteins immobilized on the surface microtiter plate wells with the retained protein being detected via ELISA. To immobilize firefly luciferase in the wells of microtiter plates, it was first chemically denatured by incubation at 5 mg/ml in 3 m guanidine HCl, 25 mm Hepes, pH 7.4, 50 mm KCl, 5 mmMgCl2, and 5 mm DTT for 1 h at room temperature. Then, 0.2 mg of denatured luciferase-made 0.1m NaHCO3 (pH 8.6) was added to wells and incubated for 30 min at 25 °C. Dot blot analysis demonstrated that under these conditions more than 90% of the added luciferase was retained in the wells. When the immobilization reaction was complete, wells were washed twice with PBS (50 mm phosphate, pH 7.4, 150 mm NaCl) and then blocked with 150 μl of 0.5% bovine serum albumin in PBS for 30 min. Wells were then washed three times with PBST (PBS containing 0.05% Tween 20). Sis1 or Sis1 mutants were then added to the wells in PBST supplemented with 0.2% BSA (PBST/BSA). After a 1-h incubation at 25 °C, the wells were washed five times with PBST. α-Sis1 rabbit polyclonal sera in 50 ml of PBST/BSA was added to the wells at a 1:5000 dilution and incubated for 1 h at 25 °C. Wells were washed five times, and then goat anti-rabbit horseradish peroxidase secondary antibody (1:5000 dilution in 50 ml PBST/BSA) was added, and incubations were carried out for 45 min. After five washes, peroxidase substrate solution was added to each well, and color formation was determined using microplate reader (Bio-Rad) set at 415 nm. Peroxidase substrate solution was prepared immediately prior to use by mixing 36 μl of 30% H2O2 and 21 ml of filtered ABTS stock solution (22 mg of ABTS/100 ml of 50 mm sodium citrate, pH 4.0). Results from control experiments demonstrated that Sis1 could be detected via ELISA over a 0.1 to 100 ng range of concentrations. In addition, we demonstrated via Western blot that all of the Sis1 mutants exhibited the same immunoreactivity to α-Sis1 as to Sis1. In experiments where reduced α-lactalbumin (LA) was utilized as the immobilized substrate of Sis1 the following protocol was employed to generate this substrate. Bovine α-LA (type III, Ca2+-depleted; Sigma) at 5 mg/ml was incubated in 10 mm DTT, 0.1 m Tris (pH 8.7), 0.2 mKCl, and 1 mm EDTA for 15 min at °C. Then 0.4 μg of reduced LA (R-LA) was added to the wells of microtiter plates in 0.1m NaHCO3 (pH 8.6) supplemented with 5 mm DTT in a volume of 50 μl. Complex formation between immobilized R-LA and Sis1 was then monitored as described above, except R-LA was maintained in its reduced stated by the addition of 2 mm DTT to all reaction mixtures. The in vivo function of theSis1 CTDI mutants was analyzed by determining whether they could support the growth of a sis1Δ strain (MATaade2-1 his3-11,15 leu2-3,112 ura3-1 trp1-1 ssd1-D2 can1-100 sis1::His3; (25Luke M.M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (147) Google Scholar) or asis1Δ::ydj1Δ strain (JJ1146;MATatrp1-1 ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 met2-D1 lys2-D2 ydj1::His3 sis1::Leu2(36Johnson J.L. Craig E.A. J. Cell Biol. 2001; 152: 851-856Crossref PubMed Scopus (85) Google Scholar). The viability of these respective strains was supported bySis1 supplied on the low copy Ura3 plasmid pRS316 (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). To swap wild-type Sis1 for its mutant forms the plasmid shuffle technique was utilized (44Boeke J.D. Trueheart J. Natsoulis G. Fink G.R. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1083) Google Scholar). The sis1Δstrain was transformed with wild-type or mutant Sis1 that was supplied on a low copy Leu2 plasmid pRS315 (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Thesis1Δ::ydj1Δ strain was transformed with wild-type or mutant SIS1 that was supplied on a low copy Trp1 plasmid pR314 (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). To counter select for theSis1 present on the Ura plasmid transformants were grown on media that contained 5-fluoroorotic acid (44Boeke J.D. Trueheart J. Natsoulis G. Fink G.R. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1083) Google Scholar). Strains were grown at 25 °C for 7 days, and the plates were then photographed. The steady state expression levels of Sis1 mutants were analyzed by Western blot of yeast extracts with a rabbit polyclonal Sis1 antibody. Freshly selected strains were grown in selective media to anA600 of 2. Yeast cells were fixed with 5% trichloroacetic acid for 5 min, and then cell pellets were twice washed with 80% acetone and resuspended in SDS-PAGE sample buffer. Lysate proteins (5 mg) were resolved on 12% SDS-PAGE and then transferred to nitrocellulose membranes. To examine the influence of wild-type Sis1 expression on the steady state level of the respective Sis1 mutants, asis1Δ strain that harbored Sis1-His6 on low copy pRS316 was generated. This strain was then transformed with wild-type and mutant forms of Sis1 on low copy pRS315 and transformants were selected on synthetic medium that was devoid of leucine and uracil. When extracts of these strains were prepared and run on 15% gels the Sis1-His6 protein migrated with a slower mobility than Sis1. This allowed for the visualization of the expression levels of non-tagged Sis1 mutants and Sis1-His6in Western blots of cell extracts. To identify regions in Sis1-(171–352) that might function in peptide-binding, GRASP analysis was utilized to probe the structure of this fragment for solvent-exposed hydrophobic residues (Fig.1, A and B) and contours (Fig. 1C). This analysis identified an unoccupied solvent-exposed patch of hydrophobic amino acid residues located on CTDI of Sis1 monomers. This patch represented the largest solvent-exposed hydrophobic region on the surface of Sis1 and is formed by residues that are contributed by β-strands 1, 2, and 5 (Fig. 1, C and D). A distinguishing feature of this patch is that it contains a 5-Å deep depression in which the solvent-exposed surface is lined by highly conserved residues that are both aliphatic and aromatic in nature (Fig. 1C). Sequence alignment of CTDI from Sis1 with similar regions from other Type II Hsp40 proteins demonstrates that residues Leu-186, Lys-199, Ile-203, and Phe-251 are 100% conserved (Fig. 1D). Whereas Val-184 and Phe-201 are found in only 20% of the Type II Hsp40s analyzed. However, in 80% of the cases a methionine residue has conservatively replaced Phe-201. Thus, CTDI of Sis1 contains a patch of solvent-exposed residues in which lies a depression that is primarily lined by conserved hydrophobic amino acids having the potential to be involved in substrate binding. To determine whether the surface-exposed residues that form the hydrophobic patch on CTDI are involved in Sis1 chaperone function, a series of point mutants was constructed (Fig.2). Then we examined the ability of purified forms of these Sis1 mutants to cooperate with Hsp70 Ssa1 in the refolding of chemically denatured luciferase (Fig. 2, Aand B). When paired with Hsp70 Ssa1, Sis1 K199A, F201H, I203T, and F251S exhibited 70–90% less folding activity than Sis1. In contrast, the protein folding activity of Sis1 V184T and L86Q was similar to that of Sis1. These data demonstrate that Lys-199, Phe-201, Ile-203, and Phe-251 are important for Sis1 to function as a co-chaperone of Hsp70 Ssa1. However, Val-184 and Leu-186 do not appear to be critical for Sis1 to function in the refolding of luciferase. For Hsp40 proteins to facilitate luciferase folding they must be able to interact with Hsp70 to stimulate its ATPase activity. To assure that the Sis1 CTDI mutants that exhibited defects in chaperone function retained the ability to interact with Hsp70, their ability to stimulate the ATPase activity of Hsp70 Ssa1 was examined (Fig. 3A). All of the Sis1 mutants tested were observed to stimulate the ATPase activity of Hsp70 Ssa1 to the same degree as Sis1. Thus, defects in regulation of Hsp70 ATPase activity do not appear to be responsible for the observed reductions in the protein folding activity of the Sis1 CTDI mutants. To rule out the possibility that mutation of CTDI caused Sis1 to misfold, thereby hindering its ability to function as a chaperone, we evaluated the folded state of the different Sis1 CTDI mutants. This was accomplished by analyzing the pattern of proteolytic fragments that were liberated by limited digestion of the respective Sis1 mutants by proteinase K. Proteinase K digestion of Sis1 generates proteolytic fragments that correspond to the J-domain and Sis1-(171–352) (Fig. 3B). When the protease resistance of purified Sis1 K199A, F201H, and I203T mutants were compared with that of Sis1, we observed no difference in the pattern of fragments formed. In contrast, Sis1 F251S was more sensitive to digestion than the other mutants. The crystal structure of Sis1-(171–352) shows that Phe-251 is located on B5 and forms the base of the depression identified in Sis1 CTDI. Phe-251 is positioned between B1 and B3 and is predicted to promote interactions between these β-strands that stabilize the Sis1 structure (37Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (145) Google Scholar). Therefore, the observation that Sis1 F251S exhibits increased sensitivity to proteinase K was not surprising. However, this result does hinder our ability to make interpretations as to whether Phe-251 is directly involved in Sis1 chaperone function or simply plays a structural role. Nonetheless, defects in the chaperone function observed for Sis1 K199A, F201H, and I203T do not appear to be a result of their defective folding. To test whether the Sis1 CTDI mutants exhibited defects in polypeptide binding, we utilized a
Referência(s)