Isolation and Characterization of a Gene Encoding a Carboxypeptidase Y-Like Protein from Arabidopsis thaliana

Abstract

Serine carboxypeptidases are proteolytic enzymes that play an important role in the intracellular turnover of proteins and in protein maturation (3, 6). In yeast and mammals, carboxypeptidase proteins have been identified that can alter another protein's activity and/or stability by removal of amino acid residues from C termini (6, 7). In plants, protein degradation is particularly important during germination, when seed storage proteins are being broken down and utilized for rapid growth. Carboxypeptidases are usually glycoproteins that are sequestered to vacuoles in plants and fungi (3, 9) and are localized to lysosomes in animals (3). Human, rat, yeast, and wheat carboxypeptidase-like genes have been isolated and their DNA sequences determined (1, 6, 7, 9). In this report, the isolation and DNA sequence determination of a carboxypeptidase Y-like gene from Arabidopsis thaliana are described. A genomic clone was isolated from a X library using a wheat cDNA clone as a hybridization probe (1). The deduced DNA sequence and carboxypeptidase Y-like protein sequence are described in Table I and Figure 1. The results of Southern blot hybridization experiments suggest that there is only one carboxypeptidase Y-like gene in the genome of Arabidopsis. All of the conserved DNA sequence features that are required for gene expression and RNA processing can be found within the DNA sequence (Table I). S 1 nuclease protection experiments were done to map the 5' end of the mRNA (Fig. 1) and to demonstrate that the cloned gene is functional. The carboxypeptidase Ylike gene consists of nine exons and eight introns. Interestingly, the deduced exon-intron boundaries are exactly conserved between the wheat and Arabidopsis genes, and five of the eight splice junctions occur between amino acid codons, whereas three junctions divide codons. Three amino acid domains have been implicated in the formation of the catalytic site of serine carboxypeptidases (24), and all three of these domains are found in the deduced Arabidopsis protein (Fig. 1, amino acids in brackets). All of the serine carboxypeptidases proteins that have been identified to date have these three amino acid domains.