Evidence for functional conservation, sufficiency and proteolytic processing of the CLAVATA3 CLE domain
Arabidopsis thaliana CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor-kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 14 amino acids termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLV3-related proteins (CLEs) in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain swap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower, Arabidopsis and BY-2 culture media that process CLV3, CLE1 and plant nematode CLEs at their C-termini. For CLV3, processing appears to occur at the highly conserved Arg70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate active CLE peptides.
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1 CLV3 and CLE proteingene information
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2h with cauliflower active CLE peptide alleles amino acids anti-GST antibodies assay AtCLE25 AtCLE4 BY-2 cells BY-2 culture media C-terminal carpels per flower cauliflower extracts CLAVATA3 CLE domain CLE family CLE motif CLE proteins CLE1 CLE6 CLE8 cleavage CLV signaling CLV1 CLV2 CLV3 CLE CLV3 expression CLV3 function CLV3 processing activity cross-complementation detected with anti-GST E.coli E.coli expressed encodes ER-CLV3 Figure fluorescence genome GST-mCLE1 GST-mCLV3 GST-tagged His-mCLV3 homozygous incubated for 2h incubated with cauliflower indicated ligand mature CLV3 mCLV3 Mean carpels mutants myc-CLV3 NCBl nematode CLE number of carpels p1 and p2 p1 fragment p1 processing Pclv3 phenotype Plant Cell polypeptide potato cyst nematode protease protein extracts proteins Proteins were separated proteolysis proteolytic proteolytic activity receptors residues SDS-PAGE and detected Selaginella separated by SDS-PAGE sequence shoot meristem signal peptide similar specific stem cell substrate suggested tissues Tobacco BY-2 cells transgene triton variable domain vitro western blotting