PLANT PHYSIOLOGY , Vol 115, Issue 4 1629-1639, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Adenosine 5[prime]-Triphosphate Is Required for the Assembly of 11S Seed Proglobulins in Vitro
Y. W. Nam, R. Jung and N. C. Nielsen
United States Department of Agriculture, Agricultural Research Service, and the Departments of Agronomy and Biochemistry, Purdue University, West Lafayette, Indiana 47907
Seed protein proglobulins were synthesized from cDNAs in reticulocyte
lysates. Most proglobulins were recovered as trimers when translation rates
were low, but mostly monomers were recovered at high translation rates. The
prevalence of monomers was accompanied by elevated amounts of insoluble
protein recovered at the bottom of sucrose density gradients. Apyrase
treatment of translation mixtures after synthesis, but before significant
assembly occurred, drastically reduced trimer assembly and increased the
proportion of insoluble aggregate. These observations indicated that ATP is
required for protein folding and/or trimer assembly. The appearance of
insoluble aggregated protein when rates of synthesis were elevated or when
ATP was absent suggested that protein misfolding had occurred. Trimer
assembly was stimulated when wheat germ translation mixtures defective in
supporting efficient trimer assembly were supplemented with fractions
isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds.
Molecular chaperones are likely involved in folding and/or assembly of
proglobulin trimers both in reticulocyte lysates and in seeds. Consistent
with this hypothesis, trimer formation was reduced when carboxymethylated
bovine albumin and [alpha]-casein, considered to mimic proteins with
extended chain and molten globular conformations and thereby compete for
Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced
into translation mixtures.
