Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls

Abstract

T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.