Molecular Cloning and Characterization of a Group II Chaperonin δ-Subunit from Soybean1.

Nong, Van Hai; Arahira, Massaomi; Van Phan, Chi; Kim, Chan-Shick; Zhang, Deyu; Udaka, Kyoko; Fukazawa, Chikafusa

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a 5-subunit homologue of CCT (CCT6). This subunit is responsible for the binding of an in vivo substrate, a-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NHj-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCT8 subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58–65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia co/i-produced soybean insoluble CCT6 as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperoning. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCT5 gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.

FORAGE plants; AMINO acids; SOYBEAN; PROTEINS; YEAST

Journal of Biochemistry, 2002, Vol 132, Issue 2, p291

0021-924X

Academic Journal

10.1093/oxfordjournals.jbchem.a003223