Redox titration of multiple protein phosphorylations in pea chloroplast thylakoids

doi:10.1016/0005-2728(93)90022-8

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Volume 1183, Issue 1, 2 November 1993, Pages 215-220

https://doi.org/10.1016/0005-2728(93)90022-8 Get rights and content

Abstract

Redox titrations were carried out on the protein kinase reactions of isolated pea chloroplast thylakoid membranes. Of the 13 phosphoproteins observed by autoradiography, all were found to titrate with the same midpoint potential of around +40 mV. Eleven proteins, including LHCII, D1, D2, CP43, a 9 kDa and a 55 kDa protein, were phosphorylated under reducing conditions, with a midpoint potential of E_m = +38 ± 4 mV, n = 0.95 ± 0.06. Two other proteins, including one at 63 kDa, were phosphorylated only under oxidizing conditions, E_m = +33 ± 11 mV, n = 0.67 ± 0.09. These midpoint potentials and n values suggest that either cytochrome b₆ or else a semiquinone associated with the cytochrome b₆f complex may be the controlling redox sensor. The ‘reverse’ redox dependence of phosphorylation of the 63 kDa and 46 kDa bands suggests that more than one redox-controlled protein kinase (or phosphatase) functions in the thylakoid membrane. We also suggest that a protein kinase (or phosphatase) is itself regulated by phosphorylation in a redox-controlled reaction.

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Cited by (35)

Abnormal Regulation of Photosynthetic Electron Transport in a Chloroplast ycf9 Inactivation Mutant
2001, Journal of Biological Chemistry
Citation Excerpt :
Importantly, this capacity to cope better with higher irradiances was not due to a more efficient PSII repair machinery since the differences in high light tolerance remained after pretreatment of leaves with lincomycin (Fig.7 B). In addition to the photoinhibition experiments, the lower phosphorylation level of PSII proteins in theycf9 mutant (Fig. 8) is also in good agreement with a considerable oxidation of PQ, since the level of PSII phosphorylation is known to respond to PQ reduction (34, 35). Notably, PSII phosphorylation in the mutant was lower also in darkness (Fig. 8).
The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadAcassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. Theycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.
Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis
1998, FEBS Letters
Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light-harvesting chlorophyll a/b proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light-intensity-dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light.
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
1997, Journal of Biological Chemistry
An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and thepsbH gene product) and the light-harvesting chlorophylla/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern 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 high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration 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 thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase.
DNA binding characteristics of CrtJ: A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus
1997, Journal of Biological Chemistry
Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatuscoded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the −10 and −35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the −10 region was calculated to be 4.8 × 10⁻⁹m, whereas affinity for the palindrome that spans the −35 region was found to be 2.9 × 10⁻⁹m. Binding of CrtJ to the bchCpromoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizingversus reducing conditions.
Effects of UVB radiation on light-dependent and light-independent protein phosphorylation in thylakoid proteins
1997, Journal of Photochemistry and Photobiology B: Biology
Protein phosphorylation in thylakoids can be divided into light-dependent and light-independent types. The phosphorylation patterns of these two types of process show about 30 and 40 phosphorylated polypeptides respectively in two-dimensional (2-D) protein separation (molecular mass discrimination in one dimension, isoelectric point (Ip) in the other). It is suggested that each type involves its own signal switch system and kinases responsible for signal transduction from the input domain to the final substrates. A thylakoid protein having a molecular mass of 45 kDa and focusing at Ip 5.0 in the 2-D protein pattern is suggested to be a kinase-like protein and its N-terminal sequence is reported. Freshly prepared spinach thylakoids were exposed to UVB (280–315 nm) radiation emitted from a 4W Philips fluorescent TL12 lamp for 60 min (0.21 W m⁻² plant weighted radiation). The proteins were extracted and subjected to 2-D gel electrophoresis. There was no distinct difference in the 2-D protein patterns between UVB-irradiated thylakoids and non-irradiated thylakoids. The experiment was repeated under conditions of light-dependent protein phosphorylation with [γ-³²P] ATP as phosphate donor. The phosphorylation of a group of polypeptides having molecular masses around 60–65 kDa and isoelectric points around 5–6, thought to include LHCII kinase, was suppressed by UVB pretreatment. The phosphorylation of LHCII polypeptides and other polypeptides was also decreased by UVB treatment. A similar experiment was performed with phosphorylation taking place in the dark after 1 h UVB (or control) treatment. In this case, the UVB treatment had an even greater impact on the phosphorylation pattern. The phosphorylation of a 45 kDa polypeptide, thought to be a kinase-like thylakoid protein active under oxidizing conditions, was almost eliminated, and that of a 60–65 kDa polypeptide group, thought to include LHCII kinase, was strongly suppressed; however, some polypeptides, e.g. 9 kDa and 10 kDa polypeptides focusing at Ip 5.7 and 7.3 respectively, became strongly phosphorylated in UVB-irradiated thylakoids. They may contribute to the protection against UVB radiation. A preliminary model is proposed for the action mechanism of both the enhanced phosphorylated proteins. We believe that altered kinase activity and protein phosphorylation may be important early steps in the impact of UVB radiation on plants.
Reply to commentary by Helmut Beinert and Patricia Kiley
1996, FEBS Letters

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¹: Permanent address: Chemistry Department D-125, Willamette University, 900 State Street, Salem, OR 97301 USA.

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Rapid reportRedox titration of multiple protein phosphorylations in pea chloroplast thylakoids

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Redox titration of multiple protein phosphorylations in pea chloroplast thylakoids