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Mutation of Residue Threonine-2 of the D2 Polypeptide and Its
Effect on Photosystem II Function in
Chlamydomonas
reinhardtii1
Christos Andronis2, *,
Olaf Kruse3,
Zsuzsanna Deák,
Imre Vass,
Bruce A. Diner, and
Peter J. Nixon
Department of Biochemistry, Imperial College of Science, Technology
and Medicine, London SW7 2AY, United Kingdom (C.A., O.K., P.J.N.); Institute of Plant Biology, Biological Research Centre, Hungarian
Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary (Z.D., I.V.); and Central Research and Development Department, Experimental Station,
P.O. Box 80173, E.I. du Pont de Nemours & Co., Wilmington, Delaware
80173-0173 (B.A.D.)
The
D2 polypeptide of the photosystem II (PSII) complex in the green alga
Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site
is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which
residue Thr-2 has been replaced by either alanine or serine. Both
mutants grew photoautotrophically at wild-type rates, and noninvasive
biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and
rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The
susceptibility of mutant PSII to photoinactivation as
measured by the light-induced loss of PSII activity in whole cells in
the presence of the protein-synthesis inhibitors chloramphenicol or
lincomycin was similar to that of wild type. These results indicate
that phosphorylation at Thr-2 is not required for PSII function or for
protection from photoinactivation. In control experiments the
phosphorylation of D2 in wild-type C. reinhardtii was
examined by 32P labeling in vivo and in vitro. No evidence
for the phosphorylation of D2 in the wild type could be obtained.
[14C]Acetate-labeling experiments in the presence of an
inhibitor of cytoplasmic protein synthesis also failed to identify
phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results
suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.
1
This research was supported by The Royal Society
(P.J.N.) and the Hungarian granting agency OTKA (nos. F017454 and
T017049). C.A. was supported by a fellowship from the State Scholarship Foundation of Greece.
2
Present address: Department of Molecular, Cell
and Developmental Biology, University of California, Los Angeles, CA
90095-1606.
3
Present address: Fakultaet Biologie,
Universität Bielefeld, 33501 Bielefeld, Germany.
*
Corresponding author; e-mail andronis{at}ucla.edu; fax
1-310-206-4386.
Plant Physiol. (1998) 117: 515-524
Copyright Clearance Center: 0032-0889/98/117/0515/10
© 1998 American Society of Plant Physiologists
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