PLANT PHYSIOLOGY , Vol 101, Issue 2 523-533, Copyright © 1993 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP, and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C
A. E. Hubbs and H. Roy
Plant Science Group, Biology Department, and Center for Biophysics, Rensselaer Polytechnic Institute, Troy, New York, 12180-3590
In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco)
consists of eight large "L" subunits, synthesized in chloroplasts, and
eight small "S" subunits, synthesized as precursors in the cytosol.
Assembly of these into holoenzyme occurs in the chloroplast stroma after
import and processing of the S subunits. A chloroplast chaperonin interacts
with the L subunits, which dissociate from the chaperonin before they
assemble into holoenzyme. Our laboratory has reported L subunit assembly
into Rubisco in chloroplast extracts after protein synthesis in leaves,
intact chloroplasts, and most recently in membrane-free chloroplast
extracts. We report here that the incorporation of in vitro-synthesized L
subunits into holoenzyme depends on the conditions of L subunit synthesis.
Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI.
When L subunit synthesis occurred at approximately 70 mM KCI, assembly
depended on the temperature at which L subunit synthesis took place. These
phenomena were the result of postsynthetic events taking place during
incubation for protein synthesis. We separated these events from protein
synthesis by lowering the temperature during protein synthesis. Lower
temperatures supported the synthesis of full-length Rubisco L subunits. The
assembly of these completed L subunits into Rubisco required intervening
incubation with ATP, before addition of S subunits. ATP treatment mobilized
L subunits from a complex with the chloroplast chaperonin 60 oligomer.
Addition of 130 mM KCI at the beginning of the intervening incubation with
ATP blocked the incorporation of L subunits into Rubisco. The inhibitory
effect of high KCI was due to CI- and came after association of newly
synthesized L subunits with chaperonin 60, but before S subunit addition.
It is interesting that L subunits synthesized at [greater than or equal
to]32[deg]C failed to assemble into Rubisco under any conditions. These
results agree with previous results obtained in this laboratory using newly
synthesized L subunits made in intact chloroplasts. They also show that
assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that
CI- inhibits Rubisco assembly, and that synthesis temperature affects
subsequent assembly competence of L subunits.
