CUCUMBER T-COMPLEX PROTEIN - MOLECULAR-CLONING, BACTERIAL EXPRESSION AND CHARACTERIZATION WITHIN A 22-S CYTOSOLIC COMPLEX IN COTYLEDONS ANDHYPOCOTYLS

T-complex protein (TCP) found in mammalian cells and yeast has been pr oposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared fr om mRNA of cotyledons of germinating cucumber seeds encodes a polypept ide 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 Ara bidopsis 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 cotyled ons and meristematic hypocotyls during seed germination. Cell fraction ations included differential centrifugation and sedimentation of large complexes at 230000Xg for 4 h. The latter fraction was further fracti onated by sedimentation velocity centrifugation. This enrichment was r equired to detect by Western blotting cytosolic 59-kDa species as cons tituents of 22-S particles. From hypocotyls, a preparation of T-comple x was obtained which consisted almost exclusively of proteins in the m olecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysat e was shown to migrate as a 61-kDa species.