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• W2126561370 abstract "The Arabidopsis BRS1 gene encodes a serine carboxypeptidase II-like protein. Its biological role in the brassinosteroid signaling pathway was first established by its capability to specifically suppress a weak brassinosteroid insensitive 1 (bri1) allele, bri1-5, when overexpressed. To gain additional insights into the molecular mechanisms of BRS1 function, the subcellular localization and the biochemical characteristics of BRS1 were determined by using transgenic plants harboring a 35S-BRS1-GFP construct and fusion proteins purified from 35S-BRS1-FLAG transgenic plants. The BRS1-GFP protein was mainly secreted and accumulated in the extracellular space. Immunological data suggest that BRS1 is proteolytically processed by an unknown endoproteinase in planta. Affinity-purified BRS1-FLAG from transgenic plants show strong hydrolytic activity with a broad P1 substrate preference including basic and hydrophobic groups on either side of the scissile bond. The hydrolytic activity of BRS1 can be strongly inhibited by a serine protease inhibitor, phenylmethylsulfonyl fluoride. The pH and temperature optima for the hydrolytic activity of BRS1 are pH 5.5 and 50 °C, respectively. These data demonstrate that BRS1 is a secreted and active serine carboxypeptidase, consistent with the hypothesis suggested by our previous genetic evidence that BRS1 may process a protein involved in an early event in the BRI1 signaling pathway. The Arabidopsis BRS1 gene encodes a serine carboxypeptidase II-like protein. Its biological role in the brassinosteroid signaling pathway was first established by its capability to specifically suppress a weak brassinosteroid insensitive 1 (bri1) allele, bri1-5, when overexpressed. To gain additional insights into the molecular mechanisms of BRS1 function, the subcellular localization and the biochemical characteristics of BRS1 were determined by using transgenic plants harboring a 35S-BRS1-GFP construct and fusion proteins purified from 35S-BRS1-FLAG transgenic plants. The BRS1-GFP protein was mainly secreted and accumulated in the extracellular space. Immunological data suggest that BRS1 is proteolytically processed by an unknown endoproteinase in planta. Affinity-purified BRS1-FLAG from transgenic plants show strong hydrolytic activity with a broad P1 substrate preference including basic and hydrophobic groups on either side of the scissile bond. The hydrolytic activity of BRS1 can be strongly inhibited by a serine protease inhibitor, phenylmethylsulfonyl fluoride. The pH and temperature optima for the hydrolytic activity of BRS1 are pH 5.5 and 50 °C, respectively. These data demonstrate that BRS1 is a secreted and active serine carboxypeptidase, consistent with the hypothesis suggested by our previous genetic evidence that BRS1 may process a protein involved in an early event in the BRI1 signaling pathway. Serine carboxypeptidases (Ser-CPs) 2The abbreviations used are: Ser-CPserine carboxypeptidaseBRbrassinosteroidCIAPcalf intestinal alkaline phosphataseFAfurylacryloylGFPgreen fluorescent proteinGUSβ-glucuronidaseLRRleucine-rich repeaterMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMES4-morpholineethanesulfonic acidMSmass spectroscopyPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideRLKreceptor-like protein kinaseRTreverse transcription. are widely distributed proteases identified in most higher organisms. The major structural characteristic of these proteins is that they contain a conserved amino acid triad, Ser-His-Asp, catalytically essential for enzyme activity (1Ramington S.J. Curr. Opin. Biotechnol. 1993; 4: 462-468Crossref PubMed Scopus (14) Google Scholar). In mammals, Ser-CPs are largely involved in producing active peptide hormones from their inactive precursors. This process usually requires two consecutive steps. First, a larger precursor is cleaved at selective sites by an endopeptidase such as prohormone convertase 1, prohormone convertase 2, or furin. The Ser-CPs are then responsible for trimming off the exposed carboxyl-terminal amino acids and transforming the inactive intermediates into active hormones (2Fan X. Olson S.J. Blevins L.S. Allen G.S. Johnson M.D. J Histochem. Cytochem. 2002; 50: 1509-1516Crossref PubMed Scopus (14) Google Scholar, 3Wei S. Feng Y. Kalinina E. Fricker L.D. Life Sci. 2003; 73: 655-662Crossref PubMed Scopus (21) Google Scholar). serine carboxypeptidase brassinosteroid calf intestinal alkaline phosphatase furylacryloyl green fluorescent protein β-glucuronidase leucine-rich repeater matrix-assisted laser desorption ionization time-of-flight 4-morpholineethanesulfonic acid mass spectroscopy phosphate-buffered saline phenylmethylsulfonyl fluoride receptor-like protein kinase reverse transcription. In plants, extensive studies of Ser-CPs have been mainly focused on their functions in turnover and mobilization of storage proteins using as nitrogen and carbon resources during seed germination and senescence (4Schaller A. Planta. 2004; 220: 183-197Crossref PubMed Scopus (313) Google Scholar). Recent studies suggested that plant Ser-CPs may also be involved in various signaling events important for plant growth and development such as programmed cell death, brassinosteroid (BR) signaling, and seed development (5Dominguez F. Cejudo F.J. Plant Physiol. 1999; 119: 81-88Crossref PubMed Scopus (42) Google Scholar, 6Li J. Lease K.A. Tax F.E. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5916-5921Crossref PubMed Scopus (187) Google Scholar, 7Cercos M. Urbez C. Carbonell J. Plant Mol. Biol. 2003; 51: 165-174Crossref PubMed Scopus (30) Google Scholar). By using a gain-of-function genetic screen we previously identified a putative Ser-CP gene, BRS1, as a bri1 (brassinosteroid insensitive 1) suppressor. Overexpression of BRS1 can suppress bri1 extracellular domain mutations but fails to suppress a kinase-dead bri1 mutant. These results strongly indicate that the bri1 suppression function of BRS1 is dependent on an at least partially functional BRI1 receptor kinase. Analyses of BRS1 protein structure and genetic data suggest that BRS1 is involved in an early step in BR signal transduction, possibly by processing a protein that may directly or indirectly participate in BR perception. As a first step toward testing this hypothesis, we tested whether BRS1 encodes an active and secreted Ser-CP. Although sequence analysis suggests BRS1 encodes a type II (D) Ser-CP, there are many enzymes related to the Ser-CPs that do not have proteolytic activity (8Dodson G. Wlodawer A. Trends Biochem. Sci. 1998; 23: 347-352Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 9Fraser C.M. Rider L.M. Chapple C. Plant Physiol. 2005; 138: 1136-1148Crossref PubMed Scopus (91) Google Scholar). In plants, these include several acyltransferases (10Li A.X. Steffens J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6902-6907Crossref PubMed Scopus (95) Google Scholar, 11Lehfeldt C. Shirley A.M. Meyer K. Ruegger M.O. Cusumano J.C. Viitanen P.V. Strack D. Chapple C. Plant Cell. 2000; 12: 1295-1306Crossref PubMed Scopus (184) Google Scholar, 12Steffens J.C. Plant Cell. 2000; 12: 1253-1256Crossref PubMed Scopus (33) Google Scholar) and a hydroxynitrile lyase (13Wajant H. Mundry K.W. Pfizenmaier K. Plant Mol. Biol. 1994; 26: 735-746Crossref PubMed Scopus (81) Google Scholar). Our previous analysis with active-site mutants of BRS1 (S181F and H438A) suggested that an active form of BRS1 is essential for its capability to suppress bri1-5, but we cannot exclude the possibility that BRS1 could be an acyltransferase rather than a Ser-CP because the proteolytic and transacylase activity of Ser-CP-like enzymes rely on the same triad of amino acids (4Schaller A. Planta. 2004; 220: 183-197Crossref PubMed Scopus (313) Google Scholar). Here we report our demonstration that BRS1 is indeed an active and secreted Ser-CP. In addition, we found that BRS1 is proteolytically processed into two chains by an unknown endoproteinase in planta. Immunopurified BRS1-FLAG protein has hydrolytic activity with a broad substrate preference. Furthermore, the hydrolytic enzyme activity can be strongly inhibited by an irreversible serine protease inhibitor, PMSF. Mutation of one of the conserved catalytic triad (H438A) of BRS1 led to inefficient protease processing and loss of the enzyme activity. These data are consistent with our previous hypothesis that BRS1 may be involved in an early proteolytic step important for BR perception and provide insight toward our understanding of plant growth and development controlled by BRs. Plant Materials and Binary Plant Transformation Vectors—Full-length BRS1 cDNA was amplified by RT-PCR and fused in-frame at its carboxyl terminus with GFP or FLAG in a pBluescript SK+-GFP and a SK+-FLAG vector. The fused BRS1-GFP and BRS1-FLAG were confirmed as in-frame and mutation-free by sequencing analyses. The inactive form of BRS1(H438A) was generated in a SK+-BRS1-FLAG construct. The fragments were removed from SK+ and cloned into pBIB-KAN and pBIB-HYG, respectively, at KpnI and SacI sites (14Becker D. Kemper E. Schell J. Masterson R. Plant Mol. Biol. 1992; 20: 1195-1197Crossref PubMed Scopus (540) Google Scholar). The fused fragments were driven by a dual enhanced CaMV35S promoter. The three transformation constructs used in these studies were designated as pKAN-35S-BRS1-GFP, pHYG-35S-BRS1-FLAG, and pHYG-35S-BRS1(H438A)-FLAG. For BRS1 expression pattern analysis, a 2-kb fragment upstream of the start codon of BRS1 was PCR-amplified and cloned into a binary vector pBIG-HYG (14Becker D. Kemper E. Schell J. Masterson R. Plant Mol. Biol. 1992; 20: 1195-1197Crossref PubMed Scopus (540) Google Scholar). The resulting construct was named pHYG-BRSp-GUS. Transgenic plants harboring 35S-BRS1-GFP, 35S-BRS1-FLAG, and 35S-BRS1(H438A)-FLAG were generated in a bri1-5 background. Transgenic plants harboring BRS1p-GUS were generated in wild type Arabidopsis (ecotype WS2) plants. In each case, the homozygous transgenic plants were selected from the T3 generation and used for the analyses described in this paper except as otherwise specified. All plants were grown in a 16-h light and 8-h dark growth chamber at 20 °C, whereas seedlings were grown under continuous light at 20 °C. RT-PCR Analyses—Total RNA was isolated using the RNeasy plant mini kit (catalog number 74904) from Qiagen (Germantown, MD). For reverse transcription, SuperScript II RNase H- reverse transcriptase from Invitrogen was used (catalog number 18064-014). Two μg of total RNA was reverse-transcribed to the first strand of the cDNA in a 20-μl volume. A 1-μl volume of the RT product was used as a PCR template. Thirty cycles were used for amplifying BRS1 and BRI1 cDNA, and 20 cycles were used for amplifying the quantity control, EF1-α. Twenty-one PCR cycles were used to compare the BRS1 expression level in inflorescence stems. GUS Staining of pHYG-BRS1p-GUS Transgenic Plants—T2 transgenic plants harboring BRS1p-GUS were used for histochemical GUS staining. Plant tissues were vacuum-infiltrated in X-Gluc solution (15Robatzek S. Somssich I.E. Plant J. 2001; 28: 123-133Crossref PubMed Scopus (333) Google Scholar) and incubated at 37 °C for 6 h followed by destaining with 70% ethanol. Confocal Microscopy—Root tips from 3-5-day-old T3 transgenic bri1-5 plants harboring 35S-BRS1-GFP were used for confocal microscopy analysis. Seeds were planted vertically on semi-solid one-half Murashige and Skoog medium (0.6% agar). Plasmolysis was induced by the addition of 0.8 m mannitol solution. Homozygous transgenic bri1-5 plants harboring 35S-BAK1-GFP were used as a positive control. Non-transformed bri1-5 plants were used as a negative control. Confocal images were obtained by using an Olympus FluoView 500 laser-scanning confocal microscope with argon laser excitation at 488 nm and 505-550 emission filter set and oil-immersion 60× objective lens. The same settings were used for all samples. Scan speed was slow, the focus mode was ×2, the dye used was enhanced GFP, and the laser output was 25 megawatts. For the enhanced GFP the photomultiplier tube voltage was 783 V, the gain was 2.6%, and offset was 5%. Western Blotting—Protein samples were run on an SDS-polyacrylamide gel and blotted to a Biotrans nylon membrane (catalog number 810300; ICN Biomedicals, Costa Mesa, CA) to immunodetect the BRS1 fusion protein. For BRS1-GFP, crude plant proteins were extracted from leaves of bri1-5 plants harboring 35S-BRS1-GFP with 2× SDS sample buffer. The primary antibody used was an anti-GFP antibody (catalog number 1814460, Roche Diagnostics). For BRS1-FLAG, the primary antibody is the anti-FLAG M2 monoclonal antibody (catalog number F3165; Sigma). The secondary antibody is goat anti-mouse IgG conjugated with horseradish peroxidase (catalog number NEF822; PerkinElmer). Signals were detected by using the Western Lighting™ chemiluminescence regent plus kit (catalog number NEL105; PerkinElmer Life Sciences). Preparation of Different Protein Fractions and Protein Immunoprecipitation—Two-week-old seedlings of homozygous bri1-5 35S-BRS1-FLAG plants were ground to a fine powder in liquid N2. The powder was further ground in cold grinding buffer (20 mm Tris-HCl (pH 8.8), 150 mm NaCl, 1 mm EDTA, 20% glycerol, 1 μm pepstatin, and 10 μm E-64). The resulting solution was spun at 6000 × g for 15 min at 4 °C, and the supernatant was collected as the total crude protein sample. The supernatant was further spun at 100,000 × g for 25 min at 4 °C. The resulting supernatant was saved as the soluble protein sample. The pellet was resuspended and further homogenized in membrane solubilization buffer (10 mm Tris-HCl (pH 7.3), 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Triton X-100, 1 μm pepstatin, and 10 μm E-64) to release membrane proteins. The solution was spun at 100,000 × g for 25 min at 4 °C to separate solubilized membrane proteins (supernatant) from the insoluble membrane fraction (pellet). Protein samples from different protein fractions were mixed with 1× PBS prewashed anti-FLAG M2 agarose affinity gel (catalog number A2220; Sigma). After overnight shaking at 4 °C, the gel was washed 5 times with cold 1× PBST (PBS and 0.1% Tween 20). Immunoprecipitated protein was eluted in 2× SDS sample buffer and run on a 7.5% SDS-polyacrylamide gel. The presence of BRS1-FLAG in different protein fractions was demonstrated by Western blotting. Protein Affinity Purification—Two-week-old seedlings of bri1-5, bri1-5 35S-BRS1-FLAG or bri1-5 35S-BRS(H438A)-FLAG were harvested and ground to fine powder in liquid N2, respectively. The suspension formed after the addition of membrane solubilization buffer as mentioned above was further homogenized to release the soluble and membrane proteins. After spinning at 100,000 × g for 25 min at 4 °C, soluble and membrane proteins were collected for affinity purification. The anti-FLAG M2 agarose affinity gel was transferred to a column (catalog number 731-1550; Bio-Rad), and the gel was washed with at least 20× volumes of 1× PBS. Then the protein samples were loaded into the column, and the flow-through was collected. Loading of the flow-through was repeated five times. The gel was washed with at least 20× volumes of 1× PBS, and finally the BRS1-FLAG was eluted with 4× 1.5-column volumes of FLAG peptide (100 ng/μl, prepared in 1× PBS; catalog number F3290, Sigma) by competition. The protein concentration was measured with a Bradford assay kit (catalog number 500-0006, Bio-Rad). Eluted protein was used for SDS-PAGE (12.5% gel) analysis and enzyme activity assay. Protein Deglycosylation and Dephosphorylation—Soluble proteins isolated from bri1-5 and bri1-5 35S-BRS1-FLAG seedlings (5 mg) were affinity-purified with anti-FLAG M2 agarose affinity gel and used for endoglycosidase H (catalog number 1088726; Roche Diagnostics Corporation) and calf intestinal alkaline phosphatase (CIAP; catalog number M1821, Promega, Madison, WI) treatment experiments. The gel was resuspended with 280 μl of water, and 15-μl aliquots of the gel were used for different treatments. Endoglycosidase treatment involved 50 mm potassium phosphate buffer (pH5.8) and 250 milliunits of endoglycosidase H incubated at 37 °C for 3 h. CIAP treatment involved 1× CIAP buffer and 10 μl of CIAP incubated at 37 °C for 2 h. In the untreated control, an equal volume of water was added instead of endoglycosidase H or CIAP. After the treatment, proteins were eluted in 2 × SDS sample buffer. Mass Spectrophotometric Assay—Affinity-purified protein with anti-FLAG M2 agarose affinity gel was run on a 12.5% SDS-polyacrylamide gel. The gel was Coomassie Blue-stained and destained in 10% ethanol and 10% acetic acid in H2O. The chosen bands were excised. In-gel trypsin digestion and MALDI-TOF mass spectrometry were conducted at the proteomics and mass spectrometry facility at the Donald Danforth Plant Science Center in St. Louis, MO. Enzyme Activity Assay—Hydrolytic activities of BRS1-FLAG toward different dipeptides were determined according to a method adapted from Plummer and Kimmel (16Plummer T.H. Kimmel M.T. Anal. Biochem. 1980; 108: 348-353Crossref PubMed Scopus (57) Google Scholar) and Latchinian-Sadek and Thomas (17Latchinian-Sadek L. Thomas D.Y. J. Biol. Chem. 1993; 268: 534-540Abstract Full Text PDF PubMed Google Scholar). Furylacryloyl (FA)-dipeptides were purchased from Bachem Biosciences Inc., King of Prussia, PA. For the time course of BRS1 hydrolytic activity, ∼400 ng of purified BRS1-FLAG protein was incubated in 1 ml of 25 mm MES (pH 5.5) containing 0.1% Triton X-100 and 1 mm FA-Arg-Leu at 37 °C for 1 h. The reduction of absorption at 342 nm was monitored every 10 min using a spectrophotometer (GENESYS 5, Spectronic Instruments, Inc. Rochester, NY). The PMSF treatment was performed as described by Latchinian-Sadek and Thomas (17Latchinian-Sadek L. Thomas D.Y. J. Biol. Chem. 1993; 268: 534-540Abstract Full Text PDF PubMed Google Scholar). Substrate, pH, and Temperature Profiles—Affinity-purified BRS1-FLAG was used for the substrate, pH, and temperature profile assays. All of the reactions except the optimal temperature experiment were done at 37 °C. The same amount of purified BRS1-FLAG and reaction buffer as described above were used for optimal substrate and temperature assays. The concentration of the substrate was 1 mm in all the reactions. The reduction of absorption at 342 nm was recorded after 1 h of incubation. One mm FA-Phe-Ala was used for the optimal pH and temperature experiments. The optimal pH for the hydrolytic activity was tested over the pH range of 4.0-7.0. The ion strength for all buffers was 25 mm. Sodium citrate buffer (pH 4.0-5.0), MES buffer (pH 5.5), and Bis-Tris buffer (pH 6.0-7.0), respectively, were used for the assays. BRS1 Exhibits a Broad Expression Pattern—brs1-1D (bri1 suppressor 1-Dominant 1) is a dominant bri1-5 suppressor identified by our previous activation-tagging genetic screen (6Li J. Lease K.A. Tax F.E. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5916-5921Crossref PubMed Scopus (187) Google Scholar). Overexpression of BRS1 can specifically suppress bri1 extracellular domain mutants instead of an intracellular kinase-dead mutant, suggesting that BRS1 may play a role in the early events of the BR signaling pathway. Because BRI1, the gene encoding the BR receptor, is expressed mainly in young tissues but less abundantly in mature tissues (18Friedrichsen D.M. Joazeiro C.A.P. Li J.M. Hunter T. Chory J. Plant Physiol. 2000; 123: 1247-1255Crossref PubMed Scopus (346) Google Scholar, 19Zhou A. Wang H. Walker J.C. Li J. Plant J. 2004; 40: 399-409Crossref PubMed Scopus (114) Google Scholar), we predict that BRS1 may have a similar or overlapping expression pattern. RT-PCR analyses indicated that BRS1 is expressed in almost all of the tissues tested (Fig. 1A). Unlike BRI1, however, BRS1 can only be detected at lower levels in roots and stems. Consistently, transgenic plants harboring BRS1p-GUS show that BRS1 is mainly expressed in young tissues such as cotyledons, young leaves, unopened flowers, and meristems (Fig. 1, B-E). These results clearly indicate that BRS1 and BRI1 have overlapping expression domains. Interestingly, we observed that most of the bri1-5 35S-BRS1-FLAG lines show ∼3-fold the height of the bri1-5 plants, whereas bri1-5 brs1-1D plants are only about two times the height of bri1-5. To test whether the BRS1 expression level is the cause of the plant height difference, total RNA was extracted from primary inflorescence stems of bri1-5, bri1-5 brs1-1D, and one representative line of bri1-5 35S-BRS1-FLAG, and RT-PCR analysis was conducted. As expected, the more the BRS1 is expressed, the taller the transgenic plants grow (Fig. 1F). These results are consistent with the idea that the reduced BR perception caused by the point mutation in bri1-5 can be partially restored by the overexpression of BRS1. BRS1-GFP Is Localized to the Exterior of the Cell—It was suggested from our previous genetic data that BRS1 may be involved in an early step in BR signaling. Because the BR perception occurs extracellularly, we predict that BRS1 should be a secreted protein. Sequence analysis indicated that BRS1 contains a typical N-terminal signal peptide but failed to identify any endoplasmic reticulum or Golgi retention signals, implying that BRS1 may be secreted from cells. To confirm this hypothesis, we generated transgenic bri1-5 plants harboring a BRS1-GFP fusion protein. These transgenic plants exhibit the bri1-5 suppression phenotype similar to that of the original bri1-5 brs1-1D suppressor and the bri1-5 35S-BRS1-FLAG transgenic plants, suggesting that the BRS1-GFP fusion protein is functionally equivalent to BRS1 in planta (Figs. 2A and 4A). Therefore, the localization revealed by BRS1-GFP should represent that of native BRS1. Using a GFP antibody to analyze the total protein extracts from the transgenic plants, several specific bands were detected. Among them, an 80-kDa band may represent the intact BRS1-GFP protein, and a 44-kDa band may represent the C-terminal BRS1 fragment attached to GFP after a predicted processing step (6Li J. Lease K.A. Tax F.E. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5916-5921Crossref PubMed Scopus (187) Google Scholar). Other smaller bands seen in the Western assay may result from partial digestion of the BRS1-GFP. Further confirmation of BRS1-GFP processing will be discussed in the latter part of this section (“Results”).FIGURE 4BRS1 protein is proteolytically processed in vivo. A, phenotypes of 3-week-old plants. Wild type BRS1-FLAG is functional in bri1-5, whereas the mutation H438A in BRS1 abolishes its function. Bar, 1 cm. B, BRS1-FLAG protein is cleaved to two chains. Protein was immunopurified from bri1-5, bri1-5 35S-BRS1(H438A)-FLAG, and bri1-5 35S-BRS1-FLAG plants. Left section shows Coomassie Blue-stained protein samples. About 300 ng of eluted protein was loaded for each lane. M, molecular mass marker; F1 and F2, two cleaved bands. Right section shows the Western blot result with anti-FLAG antibody (about one-fifth of the protein was loaded in this gel). Arrow, full-length BRS1-FLAG protein; asterisk, the C-terminal BRS1-FLAG peptide. Protein was run on a 12.5% SDS-polyacrylamide gel. C, deduced amino acid sequence of BRS1. Five tryptic peptides in a quadrupole-time-of-flight assay of F1 are shown in red. The putative cleavage linker peptide is marked as blue and italic. Arrowhead shows the predicted signal peptide cleavage site. D, MS/MS spectrum of peptide ALPGQPK in protein fragment F1. E, BRS1 is processed in vivo. Total protein was extracted with SDS buffer with (+) or without (-) β-mercaptoethanol (β-ME). Fifteen μg of protein was loaded per lane. The gel was hybridized with anti-FLAG antibody. Arrow shows chain B; triangle shows the A and B complex. F, BRS1 is not self-processed. Immunopurified BRS1-FLAG was incubated at 37 °C in 25 mm MES buffer, pH 5.5, with 0.1% Triton X-100. For the PMSF treatment, 1 mm PMSF was added to the protein and incubated on ice for 2 h before the incubation at 37 °C. About 50 ng of protein was loaded on each lane. The gel was hybridized with anti-FLAG antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Root tips from 3-5-day-old bri1-5 35S-BRS1-GFP seedlings (T3) were prepared for confocal microscopy assay. bri1-5 and bri1-5 35S-BAK1-GFP plants from similar developmental stages were used as negative and positive controls, respectively. BAK1 (BRI1-associated receptor kinase1), another crucial component in BR signal transduction pathway, was demonstrated to be a plasma membrane protein (20Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar, 21Nam K.H. Li J. Cell. 2002; 110: 203-212Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). Without any treatment, the localization of the green fluorescence signals for both BRS1-GFP and BAK1-GFP are indistinguishable, as both are apparently localized on the cell surface; after plasmolysis was induced with 0.8 m mannitol, the green fluorescence signal of BAK1-GFP moved with the plasma membrane, indicating its plasma membrane localization. The majority of the green fluorescence signal of BRS1-GFP, however, stayed in the cell wall, and only part of the green fluorescence signal moved with the plasma membrane. These observations suggest that BRS1 protein is mainly secreted and that some of the BRS1-GFP protein may associate with yet unknown membrane proteins (Fig. 2B). BRS1 Is a Glycoprotein—To further confirm that BRS1 is mainly a secreted protein and may be partially associated with membrane proteins as seen in the confocal microscopy results, the distribution of the BRS1-FLAG protein in different protein fractions was investigated. Different protein fractions were isolated from bri1-5 and homozygous bri1-5 35S-BRS1-FLAG seedlings. BRS1-FLAG is biologically functional in planta, because bri1-5 plants harboring 35S-BRS1-FLAG show a similar suppression phenotype as that of bri1-5 brs1-1D (Fig. 4A). Equal amounts of protein samples from different protein fractions were immunoprecipitated with anti-FLAG M2 agarose affinity gel overnight at 4 °C and eluted in 2× SDS sample buffer. The eluted protein samples were run on SDS-polyacrylamide gel and analyzed by Western analysis. Consistent with the subcellular localization of BRS1-GFP, Western results showed that BRS1-FLAG can be detected in both total crude and soluble protein fractions and that a small amount of BRS1-FLAG can be detected in the membrane protein fraction as well (Fig. 3A). Interestingly, there are two specific BRS1-FLAG bands revealed on the 7.5% SDS-polyacrylamide gel. The peptide mass fingerprinting analysis by MALDI-TOF mass spectrometry demonstrated that both bands are BRS1-FLAG, with the difference in migration possibly due to different post-translational modifications. To investigate whether the upper band resulted from glycosylation or phosphorylation, the soluble protein was affinity-purified and used for endoglycosidase H and CIAP treatments. Endoglycosidase H removes asparagine-linked glycosyl chains and CIAP removes phosphate. Compared with untreated BRS1-FLAG samples, only the endoglycosidase H treatment can cause the band shift of BRS1-FLAG, suggesting that BRS1 is an N-glycosylated protein but not a phosphorylated protein (Fig. 3B). After treatment, however, the double bands could still be detected. The double bands were likely the result of insufficient signal peptide removal by the endoplasmic reticulum-localized peptidase. BRS1-FLAG Is Proteolytically Processed in Vivo—Many plant serine carboxypeptidases need to be cleaved into A and B chains for activity. For example barley carboxypeptidase I is cleaved into two polypeptide chains for a heterodimer linked by disulfides, important for catalytic activity. The linker peptide containing 55 residues is endoproteolytically excised (22Doan N.P. Fincher G.B. J. Biol. Chem. 1988; 263: 11106-11110Abstract Full Text PDF PubMed Google Scholar). There is one predicted cleavage linker in the BRS1 protein sequence (6Li J. Lease K.A. Tax F.E. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5916-5921Crossref PubMed Scopus (187) Google Scholar). A Western assay suggested that BRS1 may be processed in vivo (Fig. 2A). To further confirm the in vivo processing of BRS1, large scale protein preparation and affinity purification were performed. Based on the immunological results of different protein fractions, total protein including soluble and membrane fractions was used for affinity purification. Homozygous bri1-5 35S-BRS1(H438A)FLAG transgenic plants were used as a control. To ensure the quality of the affinity purification procedure, nontransgenic bri1-5 plants were included as a negative control. In BRS1(H438A)-FLAG, one of the catalytic triad, His, was mutated to Ala. Overexpression of BRS1(H438A)-FLAG in bri1-5 failed to suppress bri1-5 defective phenotypes, which implies that BRS1(H438A)-FLAG is not biologically functional in planta (Fig. 4A). Both BRS1-FLAG and BRS1(H438A)-FLAG can be effectively purified from the transgenic plants. Typically, 9.37 μg of purified BRS1-FLAG can be recovered from 33.71 g of 2-week-old seedlings. If BRS1-FLAG is cleaved as predicted, we expect to see two bands at 34 kDa (A chain) and 19 kDa (B chain), respectively. In Fig. 4B, the left section shows the Coomassie Blue-stained high percentage SDS-polyacrylamide gel (12.5%). About 300 ng of purified protein was loaded for each sample. In the lane for bri1-5 BRS1-FLAG, besides the intact BRS1-FLAG protein band there were two ex" @default.
• W2126561370 created "2016-06-24" @default.
• W2126561370 creator A5054609598 @default.
• W2126561370 creator A5055115466 @default.
• W2126561370 date "2005-10-01" @default.
• W2126561370 modified "2023-09-27" @default.
• W2126561370 title "Arabidopsis BRS1 Is a Secreted and Active Serine Carboxypeptidase" @default.
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