Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

Complex formation in plant thylakoid membranes. Competition studies on membrane protein interactions using synthetic peptide fragments

Thylakoid membranes of pea were used to study competition between extra-membrane fragments and their parental membrane-bound proteins. Phosphorylated and unphosphorylated fragments of light harvesting complex II (LHC II) from higher plants were used to compete with LHC II for interactions with itself and with other thylakoid protein complexes. Effects of these peptide fragments of LHC II and of control peptides were followed by 80 K chlorophyll fluorescence spectroscopy of isolated thylakoids. The phosphorylated LHC II fragment competes with membrane-bound phosphoproteins in the phosphatase reaction. The same fragment accelerates the process of dark-to-light adaptation and decreases the rate of the light-to-dark adaptation when these are followed by fluorescence spectroscopy. In contrast, the non-phosphorylated LHC II peptide does not affect the rate of adaptation but produces results consistent with inhibition of formation of a quenching complex. In this quenching complex we propose that LHC II remains inaccessible to the LHC II kinase, explaining an observed decrease in LHC II phosphorylation in the later stages of the time-course of phosphorylation. The most conspicuous protein which is steadily phosphorylated during the time-course of phosphorylation is the 9 kDa (psbH) protein. The participation of the phosphorylated form of psbH in the quenching complex, where it is inaccessible to the phosphatase, may explain its anomalously slow dephosphorylation. The significance of the proposed complex of LHC II with phospho-psbH is discussed.Abbreviations LHC II light harvesting complex II - PS II Photosystem II - PS I Photosystem I     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Phosphorylation of spinach chlorophyll-protein complexes. CPII, but not CP29, CP27, or CP24, is phosphorylated in vitro

Previous studies have indicated that the reversible phosphorylation of a population of antenna complexes that can donate energy to PS II ('mobile LHC II') plays a regulatory role in the state 1-state 2 transition in thylakoid membranes. The relationship of phosphorylated LHC II to the multiple PS II-associated chlorophyll a/b-proteins resolvable on green gels is currently unclear. We have used a high resolution gel system to analyze thylakoids phosphorylated in vitro. The only PS II-associated antenna complex to become phosphorylated is CPII, indicating that this complex represents the mobile LHC II. The other putative PS II antenna complexes, CP29, CP24, and the new complex designated CP27 which comigrates with CPII, are not phosphorylated and are probably components of the bound 'LHC II' antenna.     

Light-dependent, chilling effects on phosphorylation of thylakoid proteins and consequences for associated photochemical activities in maize

When maize ( Zea mays L. cv. LG11) leaves are exposed to low temperatures and high light modifications to both photosystem 2 (PS2) and the light-harvesting chlorophyll a/b protein complex associated with photosystem 2 (LHC2) occur. This study examines the consequences of these modifications for phosphorylation of LHC2 and PS2 polypeptides and the associated changes in electron transport. Maize leaves were chilled at 5°C for 6 h under photon flux densities of 1 500 and 250 μmol m^-2 s^-1. Thylakoids were then isolated from the leaves and their abilities to phosphorylate LHC2 and PS2 polypeptides and modify electron transport activities were determined. Measurements of chlorophyll fluorescence induction in the thylakoids were also made. Thylakoids isolated from leaves chilled under high light and from leaves kept in the ambient growth environment had similar phosphoprotein profiles. However, polypeptide phosphorylation in thylakoids from the chilled leaves did not produce a decrease in PS2 electron transport. Chilling leaves under low light produced a decrease in the ability of isolated thylakoids to phosphorylate PS2, but not LHC2, polypeptides, which was not associated with any change in the phosphorylation-induced decrease in PS2 electron transport. Chilling under high, but not low, light appears to produce changes in membrane organisation that do not affect the ability of the thylakoids to phosphorylate PS2 and LHC2 polypeptides, but which do prevent the phosphorylation-induced decrease in excitation energy transfer from LHC2 to PS2.     

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

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.     

The rapidly phosphorylated 25 kDa polypeptide of the light- harvesting complex of photosystem II is encoded by the type 2 cab-II genes

The main light-harvesting complex of Photosystem II (LHC II) in higher plants consists of two sub-populations. The 'inner' pool consists only of a 27 kDa polypeptide, whereas in the 'outer' pool both the 27 kDa and a 25 kDa polypeptide are found. We purified the 25 and the 27 kDa LHC II polypeptides from Scots pine and 25 kDa LHC II polypeptide from spinach. Protein sequencing after cleavage with endoproteinase Lys-C showed that the 25 kDa polypeptide is encoded by the Type 2 cab-II genes and the 27 kDa polypeptide by the Type I cab-II genes. A fatty acid was not covalently attached to the peptides assembled into the pigment-protein complex. Our results show that the different polypeptides seen on a gel are different gene products, and not the result of different processing.     

Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes

The light-harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.     

Identification of the phosphorylation site of an 8.3 kDa protein from photosystem II of spinach

The four principal phosphoproteins of PS II cores (8.3, 32, 34 and 44 kDa) give rise to distinct tryptic phosphopeptides which have been purified by affinity chromatography on Fe³⁺-chelating Sepharose and reverse-phase HPLC. The tryptic phosphopeptide derived from the 8.3 kDa protein has the sequence NH₂-Ala-Thr-Gln-Thr-Val-Glu-Ser-Ser-Ser-Arg. It corresponds to the N-terminus of the chloroplast psbH gene product, except for the loss of the initiating N-formylmethionine. The peptide is phosphorylated on the first threonyl residue. Differences between the phosphorylation sites of the 8.3 kDa protein and LHC II are consistent with the hypothesis that thylakoids contain two distinct redox-controlled protein kinases differing in substrate specificity.     

Selective inhibition of the spinach thylakoid LHC II protein kinase

Treatment of spinach thylakoids with the adenosine affinity inhibitor 5′-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in at least 95% inhibition of phosphorylation of the light-harvesting protein complex of Photosystem II (LHC II), while the M_r 10 000 polypeptide showed a 35% decrease in phosphorylation. This residual kinase activity after FSBA treatment appears to have the same properties as the control, since phosphorylation of the M_r 10 000 polypeptide subsequent to FSBA treatment could be achieved with either light or reducing conditions in the dark. [¹⁴C]FSBA labelled several polypeptides, but only the M_r 50 000 band was protected against the label by prior addition of ADP or adenosine, making it a possible candidate for the LHC II kinase. FSBA had no effect on electron transport, and [¹⁴C]FSBA did not label LHC II or the M_r 10 000 polypeptide, indicating that the FSBA was not interfering with activation of the kinase or modifying the substrates, but rather acting at the level of the LHC II protein kinase. Inhibition of LHC II phosphorylation by FSBA resulted in the elimination of the slow ATP-induced decrease in variable fluorescence, a parameter believed to be associated with phosphorylation of the LHC II. The half-times and time-course for inhibition of LHC II phosphorylation and inhibition of the ATP-induced decrease of fluorescence yield were identical, consistent with the concept that LHC II phosphorylation plays a major role in this fluorescence change.     

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Stromal serine protein kinase activity in spinach chloroplasts

At least twelve 32P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma-32P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 °K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II → PS II energy transfer and thereby favors spillover of energy from PS II to PS I.     

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from Photosystem II

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F_v/F_m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.     

Changes in lipid composition of Photosystem 1 particles from thylakoids phosphorylated under reductive or anaerobic conditions

Changes in lipid composition of Photosystem 1 (PS 1) particles isolated from thylakoids phosphorylated under reductive or anaerobic conditions have been studied. Under reductive conditions, there was an increase in monogalactosyldiacylglycerol containing highly saturated fatty acids and phosphatidylglycerol containing transhexadecenoic fatty acid. Under anaerobic conditions, the amount of all lipid classes was increased. As we have shown earlier (S. V. Manuilskaya, O. I. Volovik, A. I. Mikhno, A. I. Polischuk and S. M. Kochubey (1990) Photosynthetica 24: 419–423) these changes were due to a co-migration of some lipid species and light-harvesting chlorophyll a/b complex LHC II from PS 2 to PS 1. These data allow us to conclude that LHC II consists of the lipoproteins containing specific lipids. Different composition of lipids co-migrating with LHC II under various conditions of phosphorylation might be caused by the variety of LHC II subpopulations transferred under each reductive condition.Abbreviations PS 1 Photosystem 1 - PS 2 Photosystem 2 - LHC II light-harvesting chlorophyll a/b protein complex II - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Identity of a Group of 13.5-20 kDa Polypeptides Whose Apparent Abundance Decreases during Plastid Development

The identity of a group of 13.5–20 kDa polypeptides whoseabundance decreases during the greening of intermittent-lightgrown barley seedlings was investigated. During greening therelative abundance of the 13.5–20 kDa polypeptides wasinversely related to that of LHC II, which had led others tosuggest a role of these polypeptides in the assembly of theLHC II and/or chloroplast development. The smallest 13.5 kDapolypeptide was identified as histone H4 by N-terminal sequencingof an internal peptide fragment produced by CNBr cleavage. Theentire group of 13.5–20 kDa polypeptides was thereafterverified to be nuclear histones by their similar mobility onSDS-PAGE to that of barley histones and immunoreactivity toyeast histone anti bodies. Their presence results from contaminationof plastid preparations by nucleosomes. Our results unequivocallysubstantiate earlier suggestions of others that polypeptidesoften found to contaminate immature plastids were of nuclearorigin. Methods to reduce or remove the histone contaminationwithout reduction in yield of thylakoids were developed so thattrue changes in the polypeptide content of thylakoids can bestudied during plastid development. (Received December 19, 1991; Accepted August 15, 1992)     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic α-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.