Ulla K. Larsson

Characterization of two different subpopulations of spinach light-harvesting chlorophyll ab-protein complex (LHC II): Polypeptide composition, phosphorylation pattern and association with Photosystem II

Abstract The properties of the light-harvesting chlorophyll a b- protein complex of Photosystem II (LHC II) was analysed in subfractions isolated from phosphorylated or heated thylakoids. The results showed that LHC II is heterogenous with respect to polypeptide composition, phosphorylation kinetics and structural association with the core of Photosystem II. One LHC II subpopulation is tightly bound to the core of Photosystem II and contains mainly a slowly phosphorylated 27 kDa polypeptide. The other subpopulation of LHC II is peripherally bound to the core and contains a relatively high proportion of a rapidly phosphorylated 25 kDa polypeptide. The latter pool of LHC II can reversibly detach from Photosystem II in respons to phosphorylation or elevated temperatures. These findings will be accommodated into a model for the dynamic arrangement of the antenna of Photosystem II.

Variations in the relative content of the peripheral and inner light-harvesting chlorophyll ab-protein complex (LHC II) subpopulations during thylakoid light adaptation and development

Abstract The light-harvesting chlorophyll a b- protein complex of Photosystem II (LHC II) is composed of two subpopulations. One population is tightly bound to the Photosystem II core and contains predominantly a 27 kDa polypeptide. The other population, enriched in a 25 kDa polypeptide, is more peripheral bound and is able to undergo reversible detachment from the core due to protein phosphorylation or moderate heat. This pool of LHC II is therefore thought to be responsible for short-term adaptation of the Photosystem II antenna. In this study we show that the peripheral subpopulation is also responsible for long-term adaptation to variations in light regime. By two-dimensional gel electrophoresis it was found that spinach or pea grown under low irradiance contained an increased amount of total LHC II and also showed an increased proportion of the 25 kDa polypeptide relative to the 27 kDa polypeptide. The additional LHC II incorporated during the low-light adaptation was calculated to possess a 27 25 kDa polypeptide ratio around 2, a ratio close to what has previously been found for the peripheral pool of LHC II. This demonstrates a specific variation in the level of the peripheral LHC II in response to long-term light adaptation. By analyzing the LHC II polypeptide composition of developing pea etioplasts, we also demonstrate that in the early stage of membrane development the Photosystem II core and its tightly bound LHC II antenna is first inserted, while the peripheral LHC II is inserted at a later stage concomitant with grana formation and segregation of the two photosystems.

Different degrees of phosphorylation and lateral mobility of two polypeptides belonging to the light-harvesting complex of Photosystem II

Abstract Several studies have shown that a subpopulation of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC-II) migrates from the appressed to the stroma-exposed thylakoids upon its phosphorylation. In this study we have analyzed the 27 and 25 kDa apopolypeptides of LHC-II, resolved by two-dimensional electrophoresis, with respect to their relative abundance and phosphorylation in thylakoids and subfractions derived from appressed or stroma-exposed thylakoid regions. The results show that the two polypeptides are heterogeneous with respect to both phosphate incorporation and degree of lateral migration. In intact thylakoids, the specific phosphorylation of the 25 kDa polypeptide exceeded that of the 27 kDa polypeptide by a factor of 3. Following phosphorylation, the 25 kDa polypeptide of the stroma lamellae showed as much as 4–5-times higher specific phosphorylation compared to the 27 kDa polypeptide. Moreover, there was a time-dependent increase in the amount of the 25 kDa polypeptide relative to the 27 kDa polypeptide in the stroma-exposed thylakoids. These results demonstrate a different polypeptide composition of the LHC-II tightly bound to Photosystem II and the free pool of LHC-II able to migrate laterally upon phosphorylation. The mobile pool of LHC-II is estimated to have two 27 kDa polypeptides for every 25 kDa polypeptide, while the ratio in the immobile pool is 4:1.

Low temperature effects on thylakoid protein phosphorylation and membrane dynamics

Abstract The effective antenna size of Photosystem II is regulated by a kinase mediated phosphorylation of the main light-harvesting chlorophyll a b protein (LHCII). This regulatory mechanism, which involves lateral migration of the phospho-protein along the thylakoid membrane, is thought to be essential for short term acclimation of the photosynthetic light harvesting. In this study we have analyzed how LHCII phosphorylation and the subsequent changes in the organization of the thylakoid membrane are influenced by low temperatures. It was shown that the kinase activity, measured as degree of LHCII phosphorylation, is operational at 0°C although partially inhibited. By subfractionation of thylakoid membranes phosphorylated at 0°C it was shown that virtually no phospho-LHCII migrates to the stroma thylakoids at this low temperature, in contrast to the case at normal temperatures. When such thylakoids, with phospho-LHCII retained in the appressed grana regions, were gradually subjected to increasing temperatures followed by subfractionation, it was shown that rapid lateral migration of phospho-LHCII was induced in a quite narrow temperature range of 10–12°C. At 5°C the migration of phospho LHCII would require hours for completion while at 20°C all phospho-LHCII had arrived in the stroma thylakoids within 5 min. From a functional point of view, our results reveal that at temperatures when the migration of phospho-LHCII from the grana region is prevented, there is no reduction in the effective antenna size of Photosystem II. This shows that protein phosphorylation in itself is not sufficient to create a functional disconnection between Photosystem II and LHCII but that the subsequent lateral diffusion of phospho-LHCII in the thylakoid membrane is required. The significance of the results in connection with increased photoinhibition during combined light and cold stress is discussed. Apart from these physiological implications, the present combination of protein phosphorylation and thylakoid subfractionation offers a novel way to study lateral diffusion of a single protein in an undisturbed biomembrane.

Heterogeneity in photosystem IIα. Evidence from fluorescence and gel electrophoresis experiments

Abstract The antenna size of different thylakoid membrane vesicles originating from grana partitions and enriched in PS IIα was determined by measuring the kinetics of the variable fluorescence and by gel electrophoresis of pigment-protein complexes. Inside-out thylakoid vesicles were fractionated by sonication and phase partition and separated into different subpopulations of PS IIα vesicles. The kinetics of the rise in the variable fluorescence of these subpopulations differed, demonstrating a heterogeneity in functional antenna size among the PS IIα. Analysis by gel electrophoresis showed that the fractions which have a more rapid rise in variable fluorescence, and hence a larger functional antenna size, also had a larger ratio of LHCII/PS II core polypeptides. The relative content of the 25 kDa polypeptide of LHCII increased with antenna size. There is thus a heterogeneity both with respect to the quantitative and the qualitative composition of the antenna of PS IIα. The results suggest that in vivo there are different domains in the partition region of the thylakoid membrane which have different average antenna size of PS IIα. The results lend further support to previous work in which heterogeneity of the antenna size of PS IIα of the same types of vesicles population was demonstrated by light saturation curves of oxygen evolution (Albertsson, P.-A. and Yu S.-G. (1988) Biochim. Biophys. Acta 936, 215–221).

Effects of bicarbonate on thylakoid protein phosphorylation

The addition of NaHCO 3 to either intact chloroplasts or isolated thylakoid membranes was found to affect thylakoid protein phosphorylation. It was found that bicarbonate highly stimulated the [ 32 P]phosphate incorporation into the 25 kDa polypepride of LHC II, whereas phosphorylation of the 27 kDa polypeptide of LHC II was unaffected. Addition of NaHCO 3 had a negative influence on the phosphorylation of the 9 kDa phosphoprotein of PS II. Moreover, depletion of endogenous bicarbonate stimulated its phosphorylation. Thesedata indicate that it is the phosphorylation of the 25-kDa- containing peripheral pool of LHC II that is under the regulation of bicarbonate. In addition, NaHCO 3 also . seems to prevent photoinhibition.

Isolation of two different subpopulations of the light‐harvesting chlorophyll a/b complex of photosystem II (LHCII)

Isolated LHCII from spinach has been solubilized and fractionated by non‐denaturing isoelectric focusing to yield two subpopulations with different polypeptide but equal chlorophyll composition. One LHCII subpopulation contains only a 27 kDa polypeptide while the other contains the 27 and 25 kDa polypeptides in about equal amounts. The polypeptide patterns of the two subpopulations closely correspond to those suggested previously for the inner LHCII and peripheral LHCII, respectively.

The 20 kDA Apo-Polypeptide of the Chlorophyll a/b Protein Complex CP24 — Characterization and Complete Primary Amino Acid Sequence

The light harvesting apparatus of higher plant photosystem II (PSII) is a highly complex and heterogenous structure consisting of several chlorophyll-binding proteins including the minor chlorophyll a/b complexes, CP29, CP27 and CP24. CP24 was first identified by Dunahay and Staehlin (1) and is estimated to contain only about 5% of the PSII chlorophyll. It is proposed to contain one (2) or several polypeptides in the range of 20–24 kDa (1). CP24 has been suggested to act as a linker protein between the light-harvesting antenna and the core of PSII (1). Others have suggested it to be associated with the light-harvesting apparatus of PSII (3,4).

The 20 kDa APO-Polypeptide of the Chlorophyll a/b Protein Complex CP24

A 20 kDa polypeptide is shown to be the only chlorophyll-binding protein of CP24. Immunoblotting of thylakoid subfractions and immunogold electron microscopy of spinach leaf sections demonstrate that CP24 is located in the PSII-rich, appressed grana regions. A cDNA clone encoding the entire precursor protein (261 aa) was isolated. The CP24 apo-poly-peptide is nuclear encoded and is predicted to have two membrane spans with putative chlorophyll binding sites in regions which shows significant homology with LHC-II.

Phosphorylation of LHCII at Low Temperatures

Higher plant thylakoids are differentiated in the lateral plane of the membrane in that photosystem II and its light-harvesting apparatus are confined to the appressed membrane regions while photosystem I and CF1/CF0 are located in the stroma exposed regions (1). However, more and more evidence are now accumulating demonstrating the dynamic nature of the thylakoid membrane. Controlled lateral movements of components between the two thylakoid regions have been reported in connection with light and heat stress (1), biosynthesis (2) and biodegradation (3). Thus, dynamic changes in the organization of the thylakoid membrane are essential for both long and short term acclimation of the photosynthetic apparatus (1).