Arabidopsis BRS1 is a secreted and active serine carboxypeptidase.
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 degrees 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.
Zhou
Aifen
A
Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019, USA.
Li
Jia
J
D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006720:Homozygote; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D019076:Transgenes
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D010455:Peptides; D049452:Green Fluorescent Proteins; D010664:Phenylmethylsulfonyl Fluoride; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; C499281:BRS1 protein, Arabidopsis