Endoproteolytic Processing of Sst2, a Multidomain Regulator of G Protein Signaling in Yeast
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m005751200
ISSN1083-351X
AutoresGinger A. Hoffman, Tiffany Runyan Garrison, Henrik Dohlman,
Tópico(s)Fungal and yeast genetics research
ResumoRegulators of G protein signaling (RGS proteins) constitute a large family of G protein-binding proteins. All RGS proteins contain a conserved core domain that can accelerate G protein GTPase activity. In addition, many family members contain a unique N-terminal domain of unknown function. Here, we demonstrate that the RGS protein in yeast, Sst2, is proteolytically processed in vivo to yield separate but functional N-terminal and RGS core domain fragments. In whole cell lysates, the full-lengthSST2 product (82 kDa) as well as a prominent 36-kDa species are specifically recognized by antibodies against the C terminus of the Sst2 protein. Purification and chemical sequencing of the 36-kDa species revealed cleavage sites after Ser-414 and Ser-416, just preceding the region of RGS homology. Expression of a mutationally truncated form of the protein (C-Sst2) could not restore function to ansst2Δ mutant strain. In contrast, co-expression of C-Sst2 with the N-terminal domain (N-Sst2) partially restored the ability to regulate the growth arrest response but not the transcription induction response. Whereas the full-length protein was localized to the microsomal and plasma membrane fractions, the N-Sst2 species was predominantly in the microsomal fraction, and C-Sst2 was in the soluble fraction. Mutations that block proteasome or vacuolar protease function, or mutations in the cleavage site Ser residues of Sst2, did not alter processing. However, Sst2 processing did require expression of other components of the pheromone response pathway, including the receptor and the G protein. These results indicate that Sst2 is proteolytically processed, that this event is regulated by the signaling pathway, and that processing can profoundly alter the function and subcellular localization of the protein. Regulators of G protein signaling (RGS proteins) constitute a large family of G protein-binding proteins. All RGS proteins contain a conserved core domain that can accelerate G protein GTPase activity. In addition, many family members contain a unique N-terminal domain of unknown function. Here, we demonstrate that the RGS protein in yeast, Sst2, is proteolytically processed in vivo to yield separate but functional N-terminal and RGS core domain fragments. In whole cell lysates, the full-lengthSST2 product (82 kDa) as well as a prominent 36-kDa species are specifically recognized by antibodies against the C terminus of the Sst2 protein. Purification and chemical sequencing of the 36-kDa species revealed cleavage sites after Ser-414 and Ser-416, just preceding the region of RGS homology. Expression of a mutationally truncated form of the protein (C-Sst2) could not restore function to ansst2Δ mutant strain. In contrast, co-expression of C-Sst2 with the N-terminal domain (N-Sst2) partially restored the ability to regulate the growth arrest response but not the transcription induction response. Whereas the full-length protein was localized to the microsomal and plasma membrane fractions, the N-Sst2 species was predominantly in the microsomal fraction, and C-Sst2 was in the soluble fraction. Mutations that block proteasome or vacuolar protease function, or mutations in the cleavage site Ser residues of Sst2, did not alter processing. However, Sst2 processing did require expression of other components of the pheromone response pathway, including the receptor and the G protein. These results indicate that Sst2 is proteolytically processed, that this event is regulated by the signaling pathway, and that processing can profoundly alter the function and subcellular localization of the protein. regulator of G protein signaling GTPase accelerating protein polyacrylamide gel electrophoresis mitogen-activated protein polymerase chain reaction 1,4-piperazinediethanesulfonic acid glutathione S-transferase nucleotide The actions of a vast array of chemical and sensory stimuli are mediated through G protein-coupled receptors. In the yeastSaccharomyces cerevisiae, the α-factor pheromone binds a receptor (Ste2), which activates a G protein and triggers a cascade of events leading to cell fusion and mating. G protein activation entails GTP binding to the α subunit (Gpa1), dissociation of Gpa1 from the βγ subunits (Ste4/Ste18), and activation of effector molecules (Ste5, Cdc24, and Ste20) that propagate the signal. Upon GTP hydrolysis, the G protein subunits reassociate and signaling stops. The RGS1 protein Sst2 attenuates G protein signaling by accelerating GTP hydrolysis and promoting subunit reassociation (1Dohlman H.G. Song J. Apanovitch D.M. DiBello P.R. Gillen K.M. Semin. Cell Dev. Biol. 1998; 9: 135-141Crossref PubMed Scopus (41) Google Scholar). RGS activity is essential for normal signal regulation in vivo. A disruption of the SST2 gene can increase pheromone sensitivity by 100–300-fold. Conversely, overexpression of SST2 can dampen the pheromone response substantially (2Dohlman H.G. Song J. Ma D. Courchesne W.E. Thorner J. Mol. Cell. Biol. 1996; 16: 5194-5209Crossref PubMed Google Scholar). The mechanism of RGS action has been well characterized through detailed biochemical and biophysical analysis of purified components (3Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 4Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 5Moy F.J. Chanda P.K. Cockett M.I. Edris W. Jones P.G. Mason K. Semus S. Powers R. Biochemistry. 2000; 39: 7063-7073Crossref PubMed Scopus (36) Google Scholar). RGS proteins act by binding and stabilizing three "switch" regions that undergo conformational change upon GTP hydrolysis. Stabilization of the transition state conformation appears to lower the energy of activation, leading to a 10–1000-fold increase in the rate of the reaction (6Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 7Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (474) Google Scholar, 8Lan K.L. Sarvazyan N.A. Taussig R. Mackenzie R.G. DiBello P.R. Dohlman H.G. Neubig R.R. J. Biol. Chem. 1998; 273: 12794-12797Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (307) Google Scholar). All RGS proteins have a common, conserved "RGS core domain" of ∼120 amino acids, which, for several RGS proteins, has been shown to be necessary and sufficient for their GTPaseaccelerating protein (GAP) activity. For instance, Wilkie and colleagues (10Popov S., Yu, K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (148) Google Scholar) have demonstrated that the RGS domains of RGS4, RGS10, and GAIP retain full GAP activity for Giα in vitro. Several other RGS proteins are considerably larger and contain additional domains or motifs that may be recognized by proteins other than Gα (11Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (228) Google Scholar, 12Siderovski D.P. Diverse-Pierluissi M. De Vries L. Trends Biochem. Sci. 1999; 24: 340-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Cabrera J.L. de Freitas F. Satpaev D.K. Slepak V.Z. Biochem. Cell Biol. 1998; 249: 898-902Google Scholar, 14Kim E. Arnould T. Sellin L. Benzing T. Comella N. Kocher O. Tsiokas L. Sukhatme V.P. Walz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6371-6376Crossref PubMed Scopus (145) Google Scholar, 15De Vries L. Lou X. Zhao G. Zheng B. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12340-12345Crossref PubMed Scopus (187) Google Scholar, 16Benzing T. Yaffe M.B. Arnould T. Sellin L. Schermer B. Schilling B. Schreiber R. Kunzelmann K. Leparc G.G. Kim E. Walz G. J. Biol. Chem. 2000; 275: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). For example, the RGS protein p115RhoGEF has one domain that acts as a GAP for G13α and a second domain that acts as a GDP-GTP exchange factor for RhoA (17Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar, 18Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (672) Google Scholar). These findings underscore the view that some RGS proteins are not simply GAPs but have separate functions that link them to other signaling pathways. Recent studies have addressed the question of how RGS proteins are themselves regulated. Several mechanisms have been established, such as alternative splicing (19Rahman Z. Gold S.J. Potenza M.N. Cowan C.W. Ni Y.G. He W. Wensel T.G. Nestler E.J. J. Neurosci. 1999; 19: 2016-2026Crossref PubMed Google Scholar), regulation of transcription (20De Vries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 21Dietzel C. Kurjan J. Mol. Cell. Biol. 1987; 7: 4169-4177Crossref PubMed Scopus (161) Google Scholar), altered localization (22Dulin N.O. Pratt P. Tiruppathi C. Niu J. Voyno-Yasenetskaya T. Dunn M.J. J. Biol. Chem. 2000; 275: 21317-21323Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Pedram A. Razandi M. Kehrl J. Levin E.R. J. Biol. Chem. 2000; 275: 7365-7372Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar), phosphorylation (25Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 26Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (40) Google Scholar, 27Benzing T. Brandes R. Sellin L. Schermer B. Lecker S. Walz G. Kim E. Nat. Med. 1999; 5: 913-918Crossref PubMed Scopus (65) Google Scholar), palmitoylation (28Druey K.M. Ugur O. Caron J.M. Chen C.K. Backlund P.S. Jones T.L. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 29Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and binding of regulatory proteins (14Kim E. Arnould T. Sellin L. Benzing T. Comella N. Kocher O. Tsiokas L. Sukhatme V.P. Walz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6371-6376Crossref PubMed Scopus (145) Google Scholar, 16Benzing T. Yaffe M.B. Arnould T. Sellin L. Schermer B. Schilling B. Schreiber R. Kunzelmann K. Leparc G.G. Kim E. Walz G. J. Biol. Chem. 2000; 275: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 30Popov S.G. Krishna U.M. Falck J.R. Wilkie T.M. J. Biol. Chem. 2000; 275: 18962-18968Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 31McEntaffer R.L. Natochin M. Artemyev N.O. Biochemistry. 1999; 38: 4931-4937Crossref PubMed Scopus (25) Google Scholar, 32Skiba N.P. Yang C.S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sst2, in particular, has been shown to be regulated by transcription and phosphorylation. SST2 mRNA levels increase by at least 5-fold in response to pheromone stimulation (21Dietzel C. Kurjan J. Mol. Cell. Biol. 1987; 7: 4169-4177Crossref PubMed Scopus (161) Google Scholar). This translates to a comparable increase in protein expression levels (2Dohlman H.G. Song J. Ma D. Courchesne W.E. Thorner J. Mol. Cell. Biol. 1996; 16: 5194-5209Crossref PubMed Google Scholar). Also in response to pheromone, Sst2 is stoichiometrically phosphorylated at Ser-539. This phosphorylation leads to an electrophoretic mobility shift, from 82 to 84 kDa, and appears to slow the overall rate of degradation of the phosphorylated 84-kDa species (25Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Here we present a novel and previously undescribed mechanism by which cells can post-translationally regulate RGS function, proteolytic processing. In the course of our studies on Sst2 phosphorylation, we noted that the mobility shift of full-length Sst2 paralleled that of a much smaller protein also recognized by our Sst2 antibodies. This smaller protein corresponds in size to the RGS core domain in Sst2 (∼36 kDa). The experiments described here were aimed at testing the possibility that this Sst2 fragment is expressed and functionalin vivo. We show that the 36-kDa product is the result of endoproteolytic processing of the full-length protein, that processing is regulated, and that this processing event leads to profound alterations in the activity and subcellular distribution of Sst2. Expression analysis was carried out in the S. cerevisiae strain YPH499 (MATa ura3-52 lys2-801 am ade2-101 oc trp1-Δ63 his3-Δ200 leu2-Δ1) or the isogenic sst2Δ strain YDM400 (YPH499, sst2-Δ2). Purification and pheromone response assays were carried out in YDM400. Signaling mutants were derived from YPH499 and are designated YDK101 (ste2::HIS3, from J. Thorner, University of California), YDM400 (2Dohlman H.G. Song J. Ma D. Courchesne W.E. Thorner J. Mol. Cell. Biol. 1996; 16: 5194-5209Crossref PubMed Google Scholar), YTG4 (ste4::hisG, this laboratory), MHY16 (ste18::LEU2) (33Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar), YTG20 (ste20::LEU2) (34Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar), YTG11 (ste11::hisG) (33Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar), YDM300 (kss1::hisG fus3::LEU2) (35Ma D. Cook J.G. Thorner J. Mol. Biol. Cell. 1995; 6: 889-909Crossref PubMed Scopus (66) Google Scholar), and YDK12/JDY3 (ste12::LEU2) (36Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar). Analysis of protease sensitivity was conducted in strains WCG4a (MATaura3 leu2-3, 112 his3-11, 15), WCG4-11/22a (WCG4a,pre1-1 pre2-2) (37Heinemeyer W. Kleinschmidt J.A. Saidowsky J. Escher C. Wolf D.H. EMBO J. 1991; 10: 555-562Crossref PubMed Scopus (357) Google Scholar, 38Heinemeyer W. Gruhler A. Mohrle V. Mahe Y. Wolf D.H. J. Biol. Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google Scholar), MHY753 (MATahis3-Δ200 leu2Δ1 ura3-52 lys2-801 trp1Δ63 ade2-101), MHY754 (MHY753, cim3-1) (39Ghislain M. Udvardy A. Mann C. Nature. 1993; 366: 358-362Crossref PubMed Scopus (370) Google Scholar), CRY1 (MATa ura3-1 leu2, 3-112 his3-11 trp1-1 ade2-1 oc can1-100), and CB007-1D (CRY1,pep4-2::HIS3 prb1::LEU2) (provided by Linda Hicke, Northwestern University). Expression plasmids used in this study are pRS315 (CEN, ampR, LEU2), pRS423 (2 μm, ampR, HIS3), pRS425 (2 μm, ampR, LEU2) (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), pRS316-ADH (CEN, ampR, URA3, ADH1 promoter/terminator) (41Song J. Hirschman J. Gunn K. Dohlman H.G. J. Biol. Chem. 1996; 271: 20273-20283Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and pAD4M (2 μm, ampR, LEU2,ADH1 promoter/terminator) (from P. McCabe, Onyx Pharmaceutical). pAD4M-SST2 was constructed by digesting SST2 withSalI (mutant site, −35 nt relative to the initiator ATG) and SacI and ligating into the corresponding sites of pAD4M (42Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (166) Google Scholar). The construction of pAD4M-SST2-his was described previously (25Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). pRS316-ADH-SST2 was constructed by digesting pRS315-GAL-SST2 (42Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (166) Google Scholar) with BamHI (mutant site, −29 nt relative to the initiator ATG; multiple cloning site adjacent to the SST2 HindIII at nucleotide 3539) and ligating into the corresponding site in pRS316-ADH. pRS316-ADH-N-SST2 was constructed by PCR so as to include a mutant BamHI site at position −1 nt with respect to the initiator ATG, SST2 codons 1–392, a Myc epitope tag (DKLDLEEQKLISEEDLLRK-STOP), and an EcoRI site three nucleotides after the stop codon. The resulting PCR product was cloned into the BamHI and EcoRI sites of pRS316-ADH. pRS315-ADH-C-SST2 (also known as ADHleu-C-SST2) was constructed by PCR so as to include a mutant BamHI site at position −1 nt with respect to Met-411, codons 411–698 of SST2, and anEcoRI site immediately following the stop codon. The PCR product was cloned into the BamHI and EcoRI sites of pRS316-ADH and then transferred as a PvuI-PvuI cassette into the corresponding sites of pRS315. pPEC-GST-SST2 was obtained from K. Madura, (Rutgers University); its construction was described previously (referred to as pPEC9) (43Schauber C. Chen L. Tongaonkar P. Vega I. Madura K. Genes Cells. 1998; 3: 307-319Crossref PubMed Scopus (19) Google Scholar). Triple substitution mutations at Ser-414, -415, and -416 were constructed in pRS316-ADH-SST2 using the QuikChange mutagenesis kit (Stratagene). The mutagenic oligonucleotides (plus complementary strands, not shown) are as follows: 5′ CT CAA GAC ATG CTT ATC GCT GCG GCT AAT TTA AAT AAG CTT GAC 3′ (Ala substitutions), 5′ CT CAA GAC ATG CTT ATC TTC TTC TTC AAT TTA AAT AAA CTG GAC 3′ (Phe), and 5′ CT CAA GAC ATG CTT ATC CAG CAG CAG AAT TTA AAT AAA CTG GAC 3′ (Gln). pRS423-FUS1-lacZ was constructed by inserting aHindIII-HindIII fragment containing theFUS1-lacZ cassette from pMD56 (44Sommers C.M. Dumont M.E. J. Mol. Biol. 1997; 266: 559-575Crossref PubMed Scopus (43) Google Scholar) into theHindIII site of pRS425 to yield pRS425-FUS1-lacZ. This product was digested with XhoI and EagI and ligated into the corresponding sites of pRS423. All PCR-amplified products were confirmed by DNA sequencing (W. M. Keck Biotechnology Resource Laboratory, Yale University). Cells were grown at 30 °C in selective media to mid-log phase and treated with 2.5 μm α-factor pheromone for 1 h, unless otherwise indicated. Temperature-sensitive mutants were grown at 24 °C to early-log phase and then shifted to 37 °C for 3 h, in the last hour they were treated with 2.5 μm α-factor. Cells were harvested by centrifugation at 2000 × g for 10 min at 24 °C, then resuspended (1.5 × 106 cells/μl) in 1× SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.0005% bromphenol blue), and boiled for 10 min. In some cases, cells were washed and stored briefly in 10 mmNaN3, on ice. The cells were disrupted by glass bead vortex homogenization (Sigma, G-8772) for 4 min and centrifuged at 16,000 × g for 2 min. The supernatant was collected and stored at −20 °C. Lysates were reheated at 37 °C for 20 min before SDS-PAGE and transfer to nitrocellulose. Immunoblots using antibodies to Sst2 (2Dohlman H.G. Song J. Ma D. Courchesne W.E. Thorner J. Mol. Cell. Biol. 1996; 16: 5194-5209Crossref PubMed Google Scholar), Pma1 (45Hager K.M. Mandala S.M. Davenport J.W. Speicher D.W. Benz Jr., E.J. Slayman C.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7693-7697Crossref PubMed Scopus (112) Google Scholar), GST (from J. Steitz, Yale University), the Myc epitope tag (46Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar), and Pgk1 (47Baum P. Thorner J. Honig L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4962-4966Crossref PubMed Scopus (50) Google Scholar) were carried out as described (48). YDM400 cells transformed with pAD4M-SST2-his were grown to A 600 nm ∼1.0, chilled, mixed with 10 mm NaN3, and harvested by centrifugation. They were then washed once in 10 mmNaN3 and rapidly frozen in liquid nitrogen. On the day of the purification, cells were thawed in Urea Buffer (6 murea, 100 mm Na2H2PO4, 10 mm Tris, and 10 mm 2-mercaptoethanol, pH 8.0), 250 mm NaCl, and 15 mm imidazole (at 250 ml per 11 liters of original culture) at room temperature. Cells were further disrupted using a stainless steel beadbeater (Biospec) packed in ice and salt, with 10 × 30-s pulses, once every 90 s. The remaining procedures were carried out at room temperature. The disrupted cells were rocked for 75 min and then clarified by centrifugation at 3840 × g for 20 min and paper filtration (Whatman No. 1). The soluble material was mixed with 3 ml of equilibrated Superflow nickel-nitrilotriacetic acid resin (Qiagen) for 60–90 min, packed into an HR 10/10 (Amersham Pharmacia Biotech) column, and washed using 10 column volumes of Urea Buffer, 250 mm NaCl, and 15 mm imidazole at 1.5 ml/min, followed by 10 column volumes of Urea Buffer at 1 ml/min. Sst2 was eluted in 10 column volumes of Urea Buffer and 75 mmimidazole at 1 ml/min. Peak fractions were pooled, concentrated, and desalted using an Ultrafree-10 (Millipore) filter. The final purified product (in 75 μl) was resolved by 11% SDS-PAGE and transferred to polyvinylidene difluoride (ProBlott, Applied Biosystems). Protein was visualized using a Coomassie Blue stain (0.025% in 50% methanol). The 36-kDa band was excised and submitted for N-terminal sequencing using an Applied Biosystems Procise 494 cLc instrument equipped with an on-line high performance liquid chromatograph (W. M. Keck Biotechnology Resource Laboratory, Yale University). Halo and reporter-transcription assays were performed as described (49Sprague Jr., G.F. Methods Enzymol. 1991; 194: 77-93Crossref PubMed Scopus (225) Google Scholar) with minor modifications. For the pheromone-dependent growth inhibition assay (halo assay), cultures were grown to saturation (2–3 days), and 100 μl was diluted with 2 ml of sterile water, followed by the addition of 2 ml of 1% (w/v) dissolved agar (60 °C). This mixture was then poured onto an agar plate containing selective medium. Sterile filter discs were spotted with synthetic α-factor pheromone (5 and 15 μg for each plate) and placed onto the nascent lawn. The resulting zone of growth-arrested cells was documented after 2 days. For pheromone-dependent reporter-transcription assays, cells were transformed with pRS423-FUS1-lacZ and grown to mid-log phase. Cultures were then aliquoted (90 μl) to a 96-well plate and mixed with 10 μl of α-factor, in triplicate. Final α-factor concentrations ranged from 0 to 100 μm. After 90 min at 30 °C, β-galactosidase activity was measured by adding 20 μl of a freshly prepared solution of 83 μm fluorescein di-β-d-galactopyranoside (Molecular Probes, 10 mm stock in Me2SO), 137.5 mmPIPES, pH 7.2, and 2.5% Triton X-100 and incubating for 90 min at 37 °C. The reaction was stopped by the addition of 20 μl of 1m Na2CO3, and the resulting fluorescence activity was measured with a multiwell plate reader using 485 nm excitation and 530 nm emission. All determinations were carried out at least twice with similar results, unless otherwise indicated. Methods for cell membrane fractionation have been described in detail elsewhere (41Song J. Hirschman J. Gunn K. Dohlman H.G. J. Biol. Chem. 1996; 271: 20273-20283Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Briefly, cells were grown at 30 °C in selective media to mid-log phase, centrifuged at 500 × g for 10 min, and resuspended in rich media (YPD) at A 600 nm = 0.5/ml. Cells were grown for one doubling period, the last hour of which they were treated with 2.5 μm α-factor. Growth was then stopped by addition of NaN3 to 10 mm. Approximately 3 × 109 cells were centrifuged and washed once with SK buffer (1.2 m sorbitol, 0.1 mKPO4, pH 7.5). Spheroplasts were then prepared by resuspending the cells in 10 ml of SK buffer containing 1 mg of zymolyase 100T (Kirin Brewery) and 28.8 mm2-mercaptoethanol for 45 min at 30 °C. All subsequent manipulations were performed at 0–4 °C. Spheroplasts were centrifuged at 500 × g for 10 min, washed once with SK buffer and once with lysis buffer (0.8 m sucrose, 20 mmtriethanolamine, pH 8, 1 mm EDTA acid, 1 mmdithiothreitol, and a protease inhibitor mixture containing 1 mm [4-(2-aminoethyl)-benzenesulfonyl fluoride, HCl] and 10 μg/ml each of leupeptin, pepstatin, and benzamidine (final concentrations)). Cell pellets were resuspended in 1 ml of lysis buffer and disrupted with 25 strokes of a motor-driven Potter-Elvehjem homogenizer. The lysate was cleared of unbroken cells and debris by centrifuging twice at 500 × g for 10 min. 200 μl of this lysate ("cleared lysate") (total, "T") was added to an equal volume of 2× SDS-PAGE sample buffer and boiled for 10 min. For isolation of total cell membranes, approximately 300 μl of the cleared lysate was centrifuged at 100,000 × g for 30 min. The top 100 μl of supernatant ("S" fraction) was diluted with 100 μl of 2× SDS-PAGE sample buffer and boiled for 10 min. The pellet ("P") was resuspended in lysis buffer to the original volume, mixed with an equal volume of 2× SDS-PAGE sample buffer, and boiled for 10 min. For resolution of cell membrane compartments, 606 mg of sucrose was added to 650 μl of the cleared lysate (T) and dissolved (final sucrose concentration 70% w/v). The sample was transferred to a Beckman thin walled polypropylene tube and overlaid with 1-ml sucrose solutions of 60, 50, 40, and 30% (w/v) in 10 mm triethanolamine, pH 8, respectively. The samples were then centrifuged in a Beckman SW40Ti swinging bucket rotor for 16 h at 190,000 × g in a Beckman L-80 ultracentrifuge. Sixteen samples of 300 μl each were collected from the bottom of the gradient into 100 μl of 4× SDS-PAGE sample buffer and boiled for 10 min. Fractions 1–14 were resolved by SDS-PAGE and immunoblotted as described above. The SST2gene encodes a 698-amino acid protein with a predicted mass of 79,696 Da. Immunoblots of whole cell lysates revealed a protein near the predicted size of the full-length product (∼82 kDa) as well as a number of prominent lower molecular weight species, one of which migrates at ∼36 kDa (Fig. 1,p36). Since our antibodies are directed to the last 365 residues of Sst2, this low molecular weight species probably corresponds to a C-terminal, proteolytically processed form of the protein. It is unlikely to be derived from another gene product, since it is absent in an SST2-deficient strain (Fig. 1,sst2Δ) and is more abundant in cells that overexpressSST2 from a plasmid (Fig. 1, sst2Δ + Sst2 o.e.). It is also not the result of alternative mRNA splicing, since SST2 is encoded by a single exon. The multiple bands in the 36-kDa region correspond to phosphorylated and unphosphorylated forms of two slightly different sized fragments of Sst2 (see below).Figure 1The RGS core domain is expressed in vivo. Whole cell lysates were prepared from theSST2-deficient strain YDM400 transformed with an empty vector pAD4M (sst2Δ), the isogenic wild-type strain YPH499 (wt strain), and strain YDM400 transformed with the Sst2-overexpression plasmid pAD4M-SST2 (sst2Δ + Sst2 o.e.). Lysates were subjected to SDS-PAGE (11% acrylamide) and immunoblotting with antibodies against Sst2 (Sst2 Ab). Specific immunoreactive bands were detected at ∼82 kDa (Full-length), 55 kDa (p55), and 36 kDa (p36). The doublets observed likely represent the phosphorylated and unphosphorylated forms of Sst2. Additional heterogeneity of the p36 species is likely due to the presence of slightly different sized Sst2 fragments (see Figs. 2 and 6). Molecular mass standards (kDa) are indicated on the left.View Large Image Figure ViewerDownload (PPT) Significantly, p36 corresponds in size to the RGS core domain of Sst2 (residues ∼417–698). For some mammalian RGS proteins, the core domain is sufficient for GTPase activating function in vitro(10Popov S., Yu, K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (148) Google Scholar), and for signal attenuating activity in cell culture (50DiBello P.R. Garrison T.R. Apanovitch D.M. Hoffman G. Shuey D.J. Mason K. Cockett M.I. Dohlman H.G. J. Biol. Chem. 1998; 273: 5780-5784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 51Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Thus we considered whether this naturally occurring fragment of Sst2 is functional as an RGS protein in vivo. We first set out to confirm the identity of the 36-kDa species. Initially, we examined whether this product extends completely to the C terminus of Sst2. Our approach here was to determine if a similarly sized fragment could be detected using an antibody directed to the extreme C terminus of the protein. Accordingly, a hexahistidine tag (−His) was appended to the 3′ end of the full-length open reading frame and expressed. The Sst2-His fusion is fully functional, as determined by its ability to complement the sst2Δ gene disruption mutant (25Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (
Referência(s)