Arabidopsis BRS1 Is a Secreted and Active Serine Carboxypeptidase

Aifen Zhou,Jia Li

doi:10.1074/jbc.m503299200

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 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.

Highlights

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 [4]
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 [4]
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

Summary

Introduction

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 [4]. 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 [8, 9]. In plants, these include several acyltransferases (10 –12) and a hydroxynitrile lyase [13]. 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

Methods

Results

Conclusion