PHOSPHORYLATION OF LIGHT-HARVESTING COMPLEX-II AND PHOTOSYSTEM-II CORE PROTEINS SHOWS DIFFERENT IRRADIANCE-DEPENDENT REGULATION IN-VIVO - APPLICATION OF PHOSPHOTHREONINE ANTIBODIES TO ANALYSIS OF THYLAKOID PHOSPHOPROTEINS
E. Rintamaki et al., PHOSPHORYLATION OF LIGHT-HARVESTING COMPLEX-II AND PHOTOSYSTEM-II CORE PROTEINS SHOWS DIFFERENT IRRADIANCE-DEPENDENT REGULATION IN-VIVO - APPLICATION OF PHOSPHOTHREONINE ANTIBODIES TO ANALYSIS OF THYLAKOID PHOSPHOPROTEINS, The Journal of biological chemistry, 272(48), 1997, pp. 30476-30482
An immunological approach using a polyclonal phosphothreonine antibody
is introduced for the analysis of thylakoid protein phosphorylation i
n vivo, Virtually the same photosystem II (PSII) core phosphoproteins
(D1, D2, CP43, and the psbH gene product) and the light-harvesting chl
orophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2),
as earlier identified by radiolabeling experiments, were recognized i
n both pumpkin and spinach leaves, Notably, the PSII core proteins and
LHCII polypeptides were found to have a different phosphorylation pat
tern in vivo with respect to increasing irradiance. Phosphorylation of
the PSII core proteins in leaf discs attained the saturation level at
the growth light intensity, and this level was also maintained at hig
h irradiances, Maximal phosphorylation of LHCII polypeptides only occu
rred at low light intensities, far below the growth irradiance, and th
en drastically decreased at higher irradiances. These observations are
at variance with traditional studies in vitro, where LHCII shows a li
ght-dependent increase in phosphorylation, which is maintained even at
high irradiances, Only a slow restoration of the phosphorylation capa
city for LHCII polypeptides at the low light conditions occurred in vi
vo after the high light-induced inactivation, Furthermore, if thylakoi
d membranes were isolated from the high light-inactivated leaves, no r
estoration of LHCII phosphorylation took place in vitro, However, both
the high light-induced inactivation and low light-induced restoration
of LHCII phosphorylation seen in vivo could be mimicked in isolated t
hylakoid membranes by incubating with reduced and oxidized diothiothre
itol, respectively, We propose that stromal components are involved in
the regulation of LHCII phosphorylation in vivo, and inhibition of LH
CII phosphorylation under increasing irradiance results from reduction
of the thiol groups in the LHCII kinase.