TRANSIENT EXPRESSION OF CLV3-GFP INDICATES RAPID INTRACELLULAR PROCESSING OF CLV3 AND A ROLE OF ENDOPLASMIN IN ITS SYNTHESIS

All plant organs are produced from pluripotent stem cells in the shoot and root apical meristems. The 12 amino acid peptide derived from the Arabidopsis thaliana soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface, in a signaling system that regulates the size of apical meristems and involves a number of receptor proteins including CLV1 and CLV2. The subcellular pathway involved in release of the CLV3 peptide from its precursor is not known. An Arabidopsis mutant expressing highly reduced amounts of endoplasmin, the endoplasmic reticulum (ER) chaperone of the HSP90 class, has a clavata-like phenotype, indicating a role of endoplasmin in the whole CLV signaling system. We show that a CLV3-GFP fusion expressed in tobacco protoplasts has very short intracellular half-life that cannot be extended by treatments with the secretory traffic inhibitors brefeldin A or wortmannin. We also show that endoplasmin specifically supports the synthesis of CLV3-GFP in an in vivo chaperone activity assay. This effect is stronger and additive to the one performed by BiP, the ER-located HSP70 chaperone. Our results indicate that processing of CLV3 starts intracellularly in early compartments of the secretory pathway and provide biochemical evidence that endoplasmin has a role in CLV3 synthesis.