The Arabidopsis AtSTE24 Is a CAAXProtease with Broad Substrate Specificity
2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês
10.1074/jbc.m202916200
ISSN1083-351X
AutoresKeren Bracha, Meirav Lavy, Shaul Yalovsky,
Tópico(s)Biofuel production and bioconversion
ResumoFollowing prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. AnArabidopsis homologue of the yeast CAAXprotease STE24 (AFC1) was cloned and expressed in rce1Δ ste24Δ mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-RacU88402fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells ofNicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation. Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. AnArabidopsis homologue of the yeast CAAXprotease STE24 (AFC1) was cloned and expressed in rce1Δ ste24Δ mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-RacU88402fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells ofNicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation. endoplasmic reticulum prenyl-dependent carboxylmethyltransferase green fluorescent protein 4-morpholineethanesulfonic acid reverse transcription wild type cyan fluorescent protein A number of eukaryotic proteins that terminate with a CXXX motif sequence are subjected to a series of post-translational modifications essential to their targeting (1Apolloni A. Prior I.A. Lindsay M. Parton R.G. Hancock J.F. Mol. Cell. Biol. 2000; 20: 2475-2487Crossref PubMed Scopus (345) Google Scholar, 2Chen Z. Otto J.C. Bergo M.O. Young S.G. Casey P.J. J. Biol. Chem. 2000; 275: 41251-41257Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 4Rodriguez-Concepcion M. Toledo-Ortiz G. Yalovsky S. Caldelari D. Gruissem W. Plant J. 2000; 24: 775-784Crossref PubMed Google Scholar, 5Rodriguez-Concepcion M. Yalovsky S. Gruissem W. Plant Mol. Biol. 1999; 39: 865-870Crossref PubMed Scopus (58) Google Scholar). The CXXX domain is usually referred to as a "CAAX box," where the C is a cysteine, the twoA residues are aliphatic amino acids, and the Xcan be one of several amino acids (6Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 30: 209-237Crossref Scopus (344) Google Scholar, 7Yalovsky S. Rodriguez-Concepcion M. Gruissem W. Trends Plant Sci. 1999; 4: 439-445Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1737) Google Scholar). The CXXX proteins undergo three sequential, enzymatic, post-translational modifications. First, the proteins are prenylated by one of two prenyltransferases called farnesyltransferase and geranylgeranyltransferase-I (6Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 30: 209-237Crossref Scopus (344) Google Scholar, 7Yalovsky S. Rodriguez-Concepcion M. Gruissem W. Trends Plant Sci. 1999; 4: 439-445Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1737) Google Scholar). Prenylation results in the covalent attachment of either farnesyl or geranylgeranyl isoprenoid lipids to the cysteine in the CAAX box motif (6Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 30: 209-237Crossref Scopus (344) Google Scholar, 7Yalovsky S. Rodriguez-Concepcion M. Gruissem W. Trends Plant Sci. 1999; 4: 439-445Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1737) Google Scholar). In yeast and animal cells, prenylation is followed by proteolytic removal of the last three amino acids of the protein (AAX) by either of the two endoproteases, RCE1 and STE24 (AFC1) (9Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar, 10Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.S. Protein Lipidation, 21. 3rd Ed. Academic Press, San Diego, CA2000: 156-213Google Scholar). This step is prenylation-dependent and is thought to take place on the cytoplasmic surface of the endoplasmic reticulum (ER) (11Schmidt W.K. Tam A. Fujimura-Kamada K. Michaelis S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11175-11180Crossref PubMed Scopus (163) Google Scholar).1 Finally, the newly exposed carboxylate group of the isoprenylcysteine is methylated by an ER-associated prenyl-dependent carboxylmethyltransferase (PCM) (10Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.S. Protein Lipidation, 21. 3rd Ed. Academic Press, San Diego, CA2000: 156-213Google Scholar, 12Clarke S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (793) Google Scholar, 13Romano J.D. Schmidt W.K. Michaelis S. Mol. Biol. Cell. 1998; 9: 2231-2247Crossref PubMed Scopus (93) Google Scholar, 14Sakagami Y. Yoshida M. Isogai A. Suzuki A. Science. 1981; 212: 1525-1527Crossref PubMed Scopus (70) Google Scholar). The CAAX proteases STE24 (AFC1) and RCE1 were first identified in a genetic screen in yeast for mutants defective in the production of a biologically active a-mating pheromone (9Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar). Farnesylation of the mating pheromone a-factor is the first in a series of modifications that convert the 36-amino acid precursor into a 12-amino acid farnesylated mature a-factor (Fig.1). Farnesylation, trimming of theAAX (VIA) moiety and carboxylmethylation are all required for the production of mature α-factor (9Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar, 15Fujiyama A. Matsumoto K. Tamanoi F. EMBO J. 1987; 6: 223-228Crossref PubMed Scopus (57) Google Scholar, 16Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). InSaccharomyces cerevisiae, STE24 (AFC1) and RCE1 act redundantly in processing the a-factor. Only RCE1, however, is responsible for processing RAS2, whereas the only known substrate of STE24 (AFC1) is the a-factor (9Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar, 17Trueblood C.E. Boyartchuk V.L. Picologlou E.A. Rozema D. Poulter C.D. Rine J. Mol. Cell. Biol. 2000; 20: 4381-4392Crossref PubMed Scopus (81) Google Scholar). Fibroblasts isolated from rce1−/− knockout mice were unable to process Ras proteins (18Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), whereas fibroblasts isolated fromzmpste24−/− knockout mice were able to process both Ras and other proteins (19Leung G.K. Schmidt W.K. Bergo M.O. Gavino B. Wong D.H. Tam A., M.N., A. Michaelis S. Young S.G. J. Biol. Chem. 2001; 276: 29051-29058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These data suggest that RCE1 may be the major functional CAAX protease in animal cells. In mice, ZmSte24 is responsible for trimming the 15 C-terminal residues of prenylated prelamin A (20Pendas A.M. Zhou Z. Cadinanos J. Freije J.M.P. Wang J. Hultenby K. Astudillo A. Wernerson A. Rodriguez F. Tryggvason K. Lopez-Otin C. Nat. Genet. 2002; 31: 94-99Crossref PubMed Scopus (423) Google Scholar). However, it has not been established whether ZmSte24 also functions as a CAAX protease of prelamin A. STE24 was initially identified as the gene responsible for processing the seven N-terminal amino acid residues of the α-factor precursor (21Fujimura-Kamada K. Nouvet F.J. Michaelis S. J. Cell Biol. 1997; 136: 271-285Crossref PubMed Scopus (131) Google Scholar). Surprisingly, STE24 and AFC1 were found to be the same gene. Further studies established that STE24/AFC1 has dual functions in α-factor maturation (22Boyartchuk V.L. Rine J. Genetics. 1998; 150: 95-101PubMed Google Scholar, 23Tam A. Nouvet F.J. Fujimura-Kamada K. Slunt H. Sisodia S.S. Michaelis S. J. Cell Biol. 1998; 142: 635-649Crossref PubMed Scopus (110) Google Scholar). AFC1 was named after first being identified as a CAAX protease. However, it was recently decided that the official of names this protein inS. cerevisiae and humans should be STE24 and Zmpste24, respectively. Thus, throughout this paper the protein will be referred to as STE24, and the Arabidopsis homologue that we identified will be referred to as AtSTE24. The prenyltransferases are soluble enzymes that are localized in the cytoplasm (7Yalovsky S. Rodriguez-Concepcion M. Gruissem W. Trends Plant Sci. 1999; 4: 439-445Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1737) Google Scholar). In yeast and animal cells, STE24, RCE1, and PCM are localized in ER membranes (11Schmidt W.K. Tam A. Fujimura-Kamada K. Michaelis S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11175-11180Crossref PubMed Scopus (163) Google Scholar, 13Romano J.D. Schmidt W.K. Michaelis S. Mol. Biol. Cell. 1998; 9: 2231-2247Crossref PubMed Scopus (93) Google Scholar, 24Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). These data suggest that CAAX proteins are prenylated in the cytoplasm and then further processed in the endomembrane system. Furthermore, in animal cells, Ha-Ras is targeted to the plasma membrane via the secretory pathway, whereas membrane targeting of K-Ras4B takes a different route (1Apolloni A. Prior I.A. Lindsay M. Parton R.G. Hancock J.F. Mol. Cell. Biol. 2000; 20: 2475-2487Crossref PubMed Scopus (345) Google Scholar, 3Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). Plant protein extracts were shown to contain PCM activity (4Rodriguez-Concepcion M. Toledo-Ortiz G. Yalovsky S. Caldelari D. Gruissem W. Plant J. 2000; 24: 775-784Crossref PubMed Google Scholar, 25Crowell D.N. Sen S.E. Randall S.K. Plant Physiol. 1998; 118: 115-123Crossref PubMed Scopus (27) Google Scholar), and a plant gene encoding PCM (AtPCM) was cloned (4Rodriguez-Concepcion M. Toledo-Ortiz G. Yalovsky S. Caldelari D. Gruissem W. Plant J. 2000; 24: 775-784Crossref PubMed Google Scholar). Transiently expressed GFP-AtPCM fusion protein accumulated in a subfraction of the endomembrane system and inhibition of the methyltransferase activity resulted in accumulation of CaM53, a prenylated plant protein, in the same subcellular fraction (4Rodriguez-Concepcion M. Toledo-Ortiz G. Yalovsky S. Caldelari D. Gruissem W. Plant J. 2000; 24: 775-784Crossref PubMed Google Scholar). Because proteolysis of the AAX moiety is a prerequisite for methylation, it was expected that CAAX proteases would be found in plants as well. However, the existence of plant homologues to either STE24 or RCE1 has yet to be reported. An important major unresolved issue in protein prenylation and related modifications in plants concerns the identification and characterization of the protease or proteases that trim the AAX group. This paper reports on the cloning and characterization ofAtSTE24, an Arabidopsis homologue of the CAAX protease STE24. AtSTE24 complementedSTE24 function in mutant yeast cells and processed a number of prenylated proteins, including a plant substrate. Searches for a homologue of RCE1 in plants, however, has so far been unsuccessful, suggesting that AtSTE24 may be the only CAAX protease in plants or that plants may have an additional as yet unknown CAAX protease. The subcellular localization of GFP-AtSTE24 was also determined. These results provide new insight into protein prenylation and the related CAAX processing events that follow it. All of the plasmids used in this study are listed in Table I.Table IPlasmids used in this studyPlasmid nameDescriptionSource or referencepJR1131Yeast low copy CEN shuttle vector containing a URA3marker, a glyceraldehydes-3-phosphate dehydrogenase gene promoter and phosphoglycerate kinase gene terminator, separated by restriction sites, AmpR gene, and bacterial origin of replicationRine laboratory and Ref. 29Yalovsky S. Trueblood C.E. Callan K.L. Narita J.O. Jenkins S.M. Rine J. Gruissem W. Mol. Cell. Biol. 1997; 17: 1986-1994Crossref PubMed Scopus (37) Google ScholarpJR1133Yeast high copy 2μ shuttle vector otherwise, identical to pJR1131Rine laboratorypJR1138Yeast high copy 2μ shuttle vector containing aLEU2 (instead of URA3) marker otherwise identical to pJR1131Rine laboratorypGFP-MRC35S∷GFP-NOS 3′ end, AmpRRef. 32Rodriguez-Concepcion M. Yalovsky S. Zik M. Fromm H. Gruissem W. EMBO J. 1999; 18: 1996-2007Crossref PubMed Scopus (130) Google ScholarpECFP-N1CFP vectorClontechpSY5535S∷CFP-NOS 3′ end, AmpRThis studypCAMBIAPlant TDNA-based binary vector, KanRCAMBIA2300pDNR-1Creator donor vector, AmpRClonthechpGEMPCR product TA cloning vector, AmpRPromegapSY16pGEM-AtSTE24This studypSY24pJR1133-AtSTE24This studypSY25pJR1131-AtSTE24This studypSY26pGFP-MRC-AtSTE24This studypSY27pCAMBIA 2300-GFP-AtSTE24This studypSY58pCFP-AtSTE24This studypSY59pCAMBIA2300-CFP-AtSTE24This studypSY77pDNR-1-AtSTE24This studypSY78pDNR-1-Atste24mA284This studypSY62pJR1133-Atste24mA284This studypSY99pGFP-Racu88042mSThis studypSY110pGFP-RacU88042This studypSY29pJR1138 GFP-Racu88042mSThis studypSY30pJR1138 GFP-RacU88042This studypSY56pGFP-AtBDCaM53Ref.4Rodriguez-Concepcion M. Toledo-Ortiz G. Yalovsky S. Caldelari D. Gruissem W. Plant J. 2000; 24: 775-784Crossref PubMed Google ScholarpSY86pJR1138 GFP-AtBDCaM53This studypSY106pGEM RacU88042This studypSY107pGEM Racu88042mSThis studyp35S-ACA2-GFPPlant TDNA binary vector expressing the ER marker ACA2pRef. 31Hong B. Ichida A. Wang Y. Scott J.S. Pickard B.G. Harper J.F. Plant Physiol. 1999; 119: 1165-1175Crossref PubMed Scopus (103) Google Scholar Open table in a new tab The protein data base was searched for a plant homologue of STE24 using the yeast and human STE24 amino acid sequences. A genomic sequence from Arabidopsis thaliana(GenBankTM accession number AF007269), encoding for a putative protein showing 50% homology to both yeast and human STE24 proteins, was identified. A 1275-bp fragment was amplified from a flower cDNA library using oligonucleotide primers P1 (5′-TAGAGGATCCAGATGGCGATTCCTTTCATGGAAACC-3′) and P2 (5′-ACCGCTCGAGTCTAGATTAATCTGTCTTCTTGTCTTCTC-3′). The amplified fragment was subcloned into pGEM (Table I) to create pSY16. pSY16 was then used to fully sequence AtSTE24 cDNA. To express the GFP-AtSTE24 fusion protein in plants, aBamHI-XbaI fragment containing a full-lengthAtSTE24 cDNA sequence was cloned into pGFP-MRC (Table I) in frame with the C terminus of GFP to create pSY26. Subsequently, pSY26 was digested with SphI to isolate a 3.5-kb fragment containing the cauliflower mosaic virus (CaMV35S) promoter, GFP-AtSTE24, and NOS 3′-end, which was subcloned into the plant binary vector, pCAMBIA2300, to create pSY27 (Table I). To express AtSTE24 inS. cerevisiae, a BamHI-SalI fragment of AtSTE24 was subcloned into the yeast plasmids, pJR1133 and pJR1131 (Table I), to create plasmids pSY24 and pSY25, respectively. An expressed sequence tag of the Rac-like U88402cDNA (GenBankTM accession number U88402) was identified by data base searches for CAAX motif plant proteins. Clone number D20T7, containing RacU88402, was obtained from theArabidopsis Biology Resource Center. Two oligonucleotide primers were designed to place appropriate restriction sites on the 5′- and 3′-ends: P129 (5′-CATATGGAGCTCGCAGATGCAAACGAAGTAGTG-3′) and P124 (5′-CCGCTCGAGTCTAGATTACATCAACAAGCAGCGACG-3′). An 806-bp fragment was amplified by PCR and cloned into pGEM to create pSY106. A racU88402mS mutant in which the prenyl acceptor cysteine residue had been changed to serine was created by PCR using P129 and p125 (5′-CCGCTCGAGTCTAGATTACATCAACAAGCTGCGACGCTC-3′) oligonucleotide primers. The resulting fragment was cloned into pGEM to create pSY107. To express RacU88402 and racU88402mS fused to GFP, SacI-XbaI fragments of U88402 and u88402mS cDNAs were cloned into pGFP-MRC (Table I) in frame with the C terminus of GFP to create pSY110 and pSY99, respectively. To express GFP-U88402 and GFP-u88402mS in S. cerevisiae, pSY99 and pSY110 were digested with XhoI and XbaI to isolate GFP-U88402 and GFP-u88402mS fragments, which were subsequently blunt-ended with Klenow DNA polymerase and subcloned into pJR1138 (Table II).Table IIYeast strains used in this studyStrainGenotypeSource or referenceJRY 6958MATa his3 leu2 met15 ura3 pep4Δ∷KanMXRine laboratoryJRY 6959MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3Rine laboratoryJRY 6961MATα rce1Δ∷TRP his3 leu2 lys2 met15 ura3 pep4Δ∷KanMXRine laboratoryJRY 6962MATα ste24Δ∷HIS his3 leu2 met15 ura3Rine laboratoryJRY 3443MATα sst2 trp1 his3 ura3 can1Rine laboratorySYY 500MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pSY24Transformant of JRY 6959SYY 501MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pJR1133Transformant of JRY 6959SYY 502MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pSY25Transformant of JRY 6959SYY 503MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pJR1131Transformant of JRY 6959SYY 508MATa his3 leu2 met15 ura3 pep4Δ∷KanMX + pSY30Transformant of JRY 6958SYY 509MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pSY30Transformant of JRY 6959SYY 510MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pSY 30 + pSY24Transformant of SYY 500SYY 515MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 ura3 + pSY24 + pSY86Transformant of SYY 500SYY 516MATa his3 leu2 met15 ura3 pep4Δ∷KanMX + pSY86Transformant of JRY 6958SYY 517MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 + pSY86Transformant of JRY 6959SYY 518MATα rce1Δ∷TRP his3 leu2 lys2 met15 ura3 pep4Δ∷KanMX + pSY30Transformant of JRY 6961SYY 519MATα ste24Δ∷HIS his3 leu2 met15 ura3+ pSY30Transformant of JRY 6961SYY 520MATa ste24Δ∷HIS rce1Δ∷TRP his3 leu2 lys2 met15 + pSY62Transformant of JRY 6959 Open table in a new tab Although Rac88402 was obtained from the Arabidopsis Biology Resource Center as an expressed sequence tag clone, no genomic homologue could be identified in the Arabidopsis data base. All our attempts to amplify RacU88402, either directly fromArabidopsis cDNA libraries or by RT-PCR, proved fruitless. Rac88402 shows the highest homology to a Rac2 protein ofDictyostelium discoideum, and curators at theArabidopsis data base have suggested that its likely origin is contamination of the expressed sequence tag library with foreign RNA. All of the clones were fully sequenced to confirm that no PCR and cloning-generated errors had been introduced. All of the yeast strains used in this study are listed in Table II. Yeast transformation was carried out with a standard lithium acetate transformation protocol (26Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). The assays were carried out essentially as described previously (17Trueblood C.E. Boyartchuk V.L. Picologlou E.A. Rozema D. Poulter C.D. Rine J. Mol. Cell. Biol. 2000; 20: 4381-4392Crossref PubMed Scopus (81) Google Scholar) with the following modifications. Three μl of MATa cell slurry (∼107 cells) were spotted onto a solid, rich medium (YPD) plate containing 0.01% Triton X-100 that had been spread with a lawn (∼2 × 106 cells) of the MATα sst2cells (JRY3443). After 1 day of growth at 28 °C, the relative amounts of α-factor produced by each MATa strain were evident from the size of the growth inhibition zone (halo) surrounding the MATa cells. The relative halo sizes were directly proportional to the amount of α-factor exported from theMATa cells (27Reneke J.E. Blumer K.J. Courchesne W.E. Thorner J. Cell. 1988; 55: 221-234Abstract Full Text PDF PubMed Scopus (239) Google Scholar). Nicotiana benthamiana plants were grown in 10-cm pots. The seeds were sown on a mix of 70% soil with vermiculite (Avi Saddeh mix, Pecka Hipper Gan) and 30% sea sand and were irrigated from below. The plants were grown in an environmental growth chamber under long days (16 h light/8 h dark cycles) at 27 °C. Light intensity was 100 μE m−2s−1. Young leaves from N. benthamianawere injected with Agrobacterium tumefaciensGV3101/mp90 strains harboring plasmids pSY27 and pSY59. Ten-ml cultures were grown overnight at 28 °C in LB liquid medium with kanamycin (50 μg/ml). The next day, cultures were harvested by centrifugation and resuspended in 4 volumes of induction medium (50.78 mm MES, 0.5% glucose, 1.734 mm NaH2PO4, 0.2 mm acetosyringone, and 5% 20X-AB mix (20X-AB mix comprised 373.9 mm NH4Cl, 24.34 mmMgSO4, 40.23 mm KCl, 1.36 mmCaCl2, 0.18 mmFeSO4-7H2O)). The cells were grown for an additional 6 h until A600 = 0.2–0.8. The cultures were then diluted with induction medium to A600 = 0.2 and injected into the abaxial leaf side using a 1-ml syringe (without a needle). To inject, the syringe was held against the surface of a leaf while applying counter-pressure with a finger to the opposite (adaxial) surface. The leaves were observed for GFP fluorescence at 12–24 h post-injection. Wide field fluorescence imaging was carried out on a Zeiss Axioplan-2 Imaging fluorescent microscope, with an AxioCam cooled CCD camera, a Zeiss filter set 40, and the appropriate Pinkel set filters. Confocal laser fluorescence imaging was carried out using a Zeiss R510 confocal laser scanning microscope. Excitation was carried out with an Argon laser set to 488 nm. Emission was detected with a 525 ± 15 nm band path filter. Zeiss AxioVision, Zeiss CLSM-5, and Adobe Photoshop 6.0 were used for image analysis. Real time RT-PCR was performed in a fluorescence temperature cycler (LightCycler; Roche Molecular Biochemicals). Total RNA was isolated from various tissues using an SV total RNA isolation Kit (Promega). cDNA first strand synthesis was performed as previously described (28Caldelari D. Sternberg H. Rodriguez-Concepcion M. Gruissem W. Yalovsky S. Plant Physiol. 2001; 126: 1416-1429Crossref PubMed Scopus (48) Google Scholar). cDNA corresponding to 50 ng of RNA served as a template in a 20-μl reaction containing 4 mm MgCl2, 0.5 mm gene specific primers, and 2 μl of LightCycler-FastStart DNA Master SYBR Green-I mix (Roche Molecular Biochemicals). The samples were loaded into capillary tubes and incubated in the fluorescence thermocycler (LightCycler) for an initial denaturation at 95 °C for 10 min. The PCR reaction consisted of 45 cycles of 15 s at 95 °C, 10 s at 55 °C, and 55 s at 72 °C. The amount of PCR products was estimated by measuring SYBR Green-I fluorescence at the end of each cycle. To confirm amplification of specific transcripts, melting curve profiles were produced at the end of each run. These melting curves were produced by measuring the fluorescence of samples cooled to 65 °C for 25 s and then reheated to 95 °C at increments of 0.1 °C/s. A 1273-bp fragment of AtSTE24 was amplified with primers p1 (5′-TAGAGGATCCAGATGGCGATTCCTTTCATGGAAACC-3′) and p2, (5′-ACCGCTCGAGTCTAGATTAATCTGTCTTCTTGTCTTCTC-3′). A 520-bp fragment of ubiquitin was amplified with primers: CGATTACTCTTGAGGTGGAG and AGACCAAGTGAAGTGTGGAC. Cycle-to-cycle fluorescence emission readings were monitored and analyzed using LightCycler Software (Roche Molecular Biochemicals). The software first normalizes each sample by detecting the background fluorescence present in the initial cycles. 5% of the full scale fluorescence threshold is then set, and the software determines the cycle number at which each sample reaches this threshold. The cycle number at which this 5% threshold is reached correlates inversely to the log of the initial template concentration. Relative levels ofAtSTE24 transcript were corrected by normalization against the ubiquitin transcript levels. The specificity of the amplification products was further verified by subjecting the amplification products to electrophoresis on a 1% agarose gel. Yeast strain SYY 515 (ste24Δ rce1Δ expressing GFP-CaM53), SYY 500, JRY 6958, and JRY6959 (Table II) were grown in minimal medium with the appropriate supplements to A600 = 0.7. Total protein extracts were prepared as described previously (29Yalovsky S. Trueblood C.E. Callan K.L. Narita J.O. Jenkins S.M. Rine J. Gruissem W. Mol. Cell. Biol. 1997; 17: 1986-1994Crossref PubMed Scopus (37) Google Scholar). Methylation assays were carried out as follows: 20 μl of SYY 515 protein extract were incubated with 20 μl of total protein extracts prepared from either SYY 500, JRY 6958, or JRY6959 in the presence of a reaction buffer containing 100 mm Hepes-KOH, pH 7.4, 5 mm MgCl2, 50 μmZnCl2, 1 mm phenylmethylsulfonyl fluoride, 1 μCi of [3H]AdoMet (69 Ci/mmol), and 0.5 μm AdoMet. The reactions were carried out at 30 °C for 30 min. The reactions were terminated by heat denaturation in an SDS sample buffer (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). Aliquots (15 μl) of each reaction were fractionated in turn on SDS-PAGE (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). The proteins were electrotransferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were decorated with mouse anti-GFP monoclonal antibodies (Stress-Gene) as primary antibodies, followed by blotting grade goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). The blots were developed with Super Signal Substrate kit (Pierce) and exposed to x-ray film (Kodak XAR). To determine the methylation of GFP-CaM53, the x-ray films were placed on the nitrocellulose membranes, and the corresponding bands were excised out of the membranes and incubated in scintillation liquid for 8 h prior to counting. To ensure that all of the counts were GFP-CaM53-dependent, corresponding membrane strips that did not contain GFP-CaM53 were excised and counted. These strips gave counts of 10–30 cpm. These background counts were subtracted from all GFP-CaM53-dependent counts. The reactions were carried out in triplicate, and the results are presented as the average counts obtained relative to wt RCE STE24, which were taken as 100%. ABamHI-XbaI fragment containing a full-length AtSTE24 cDNA sequence was subcloned into pDNR-1 in frame with the lox-p site to create pSY77 (pDNR-1-AtSTE24) (Table I). pSY77 was used as the template together with primers p14 (5′-GTGGCGGTTATTGCAGCCGAGCTTGGACATTGG-3′) and p15 (5′-CCAATGTCCAAGCTCGGCTGCAATAACCGCCAC-3′) to mutate AtSTE24 His284 to alanine using a QuikChange site-directed mutagenesis kit (Stratagene) to create pSY78. Atste24mA284was sequenced to verify that no PCR generated errors were introduced. pSY78 was digested with BamHI and XbaI to isolate a Atste24mA284 fragment, which was blunt-ended with Klenow fragment DNA polymerase and subcloned into pJR1133 to create pSY62. Yeast and human STE24 amino acid sequences were used to search theArabidopsis data base. A genomic sequence of 3097 bp from chromosome 4 (GenBankTM accession number AF007269) with a deduced 939-bp cDNA showing high homology to both yeast and human STE24 was identified. We termed this gene AtSTE24. AtSTE24 cDNA was cloned from a flower cDNA library using a PCR-based approach with oligonucleotide primers, designed according to the genomic sequence. The PCR amplification yielded a 1275-bp fragment, which was 336 bp longer than the sequence predicted by the Arabidopsis Genome Initiative. Using the same primers in an RT-PCR reaction, a 1257-bp fragment was also amplified from a Col-0 poly(A)+ RNA (se
Referência(s)