Separation of subchloroplast membrane particles by counter-current distribution.

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.     

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity.The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique.Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks.The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.     

Analysis of the polypeptide composition of grana and stroma thylakoids by two-dimensional gel electrophoresis. Distribution of photosystem II between grana and stroma lamellae

The polypeptide composition of whole thylakoids and membrane subfragments was studied by using a modified two-dimensional gel electrophoresis technique of O'Farrell [J. Biol. Chem. 250, 4007-4021 (1975)]. The modifications were lithium dodecyl sulphate solubilization instead instead of SDS, reverse isofocusing and sensitive silver staining procedure. This high-resolution technique allowed us to separate and identify about 170 polypeptides of thylakoid membranes. After separating grana and stroma thylakoids it was found that both types of lamellae contained nearly equal amounts of polypeptides, but about 70 polypeptides were different in the two preparations. In grana thylakoids, 54 polypeptides out of 95 were found to be mainly present in grana and 31 of them were only present in grana preparations. In stroma membranes, 43 polypeptides out of 99 were mainly present in stroma lamellae and 38 of these polypeptides were exclusively present in stroma lamellae. In a functional photosystem II preparation, 61 individual polypeptides could be distinguished. Most of these polypeptides were present in both grana and stroma lamellae, but 22 of them were more pronounced in grana than in stroma lamellae. 9 polypeptides of photosystem II were distinctly different in grana and stroma lamellae, and these differences may connect closely with the functional differences of photosystem II in the two types of thylakoids.     

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound.

The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex.

In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10–20% of the total Photosystem II reaction centre complex and the light-harvesting complex.

The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex.

The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.     

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer.

The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H₂O to phenyl-p-benzoquinone and ascorbate-Cl₂Ind to NADP⁺ electron transport rates indicate a ratio of around six between Photosystem II and I.

The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP⁺.

These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

The polypeptides of stacked and unstacked Chlamydomonas reinhardi chloroplast membranes and their relation to photosystem II activity

When cells of the ac-5 mutant strain of Chlamydomonas reinhardi are cultured mixotrophically, their chloroplast membranes are unstacked and they lack a group of membrane polypeptides that have been reported to be associated with a membrane fraction enriched for Photosystem II activity. On the other hand, the chloroplast membranes of cells grown phototrophically are stacked and they possess the membrane polypeptides. Since the unstacked membranes possess Photosystem II activity, we suggest that the polypeptides must be present in the chloroplast membrane if stacking is to occur.     

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.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley

Chlorophyf l-free preparations of plasma membranes from leaves of barley (Hordeum vulgare L. cv. Kristina) and spinach (Spinada oleracea L. cv. Viking II) were obtained by partition in an aqueous dextran-polyethylene glycol two-phase system. CJlu-can synthetase II (EC 2.4,1.34), a marker for the plasma membrane, was highly enriched in both preparations. Silicotungstic acid, a specific stain for the plasma membrane, indicated a purity close to 100% for the barley preparation. Both plasma membrane preparations contained a light-reducible b-cytochrome, as shown by low temperature spectroscopy. The plasma membranes had a tow protein content compared to the bulk of intracellular membranes. The polypeptide composition of the barley and spinach plasma membranes showed striking similarities, with.the most prominent polypeptides in the 49-58 kdalton region, and some further prominent bands in the 30 kcialton region. Some high molecular weight polypeptides in the 73-110 kdalton region were also typical for the plasma membranes compared to the microsomal fractions.     

Membrane polypeptides of some higher plant chloroplasts

Sodium dodecylsulfate-polyacrylamide gel electrophoresis of membrane polypeptides of the mesophyll cell chloroplasts of barley, pea, and maize show similar profiles, with the polypeptides falling into two major groups: those associated with a membrane fraction enriched in Photosystem I (called Group I polypeptides) and those associated with a membrane fraction enriched in Photosystem II (called Group II polypeptides a, b, and c). In contrast to these profiles, the polypeptides from the extensively unstacked membranes of chloroplasts from the chlorophyll-deficient mutant strains of barley and pea as well as those obtained from the agranal bundle sheath cell chloroplasts of maize are deficient in the Group II polypeptides b and c. It is proposed that these polypeptides are required for membrane stacking in higher plant chloroplasts.These Group II polypeptides b and c are not required for Photosystem II activity since both the barley and pea mutant chloroplasts and the maize bundle sheath chloroplasts possess Photosystem II activities.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

Fat metabolism in higher plants. LVI. Distribution and nature of biotin in chloroplasts of different plant species

A number of bacteria, algae, and higher plant chloroplasts were examined to determine the nature of their biotin-protein complexes. In all tissues studied, the major fraction of the total biotin was bound to protein(s) through a lysine bridge and these proteins accepted ¹⁴CO₂ to form carboxybiotinyl protein(s). The biotinyl protein was present in the soluble protein fraction in the procaryotic organisms, Escherichia coli and Rhodospirillum rubrum. In eucaryotic organisms, such as Chlamydomonas reinhardi and chloroplasts from higher plants, biotinyl protein was associated with chloroplast membranes. The blue-green alga, Anacystis nidulans, showed an intermediate condition, while the filamentous blue-green alga, Anabaena flos-aquae, resembled the higher plant chloroplasts. Although on a chlorophyll basis, stroma lamellae fractions enriched in Photosystem I had a higher biotin protein content than did the grana lamellae fractions, on a protein basis, the biotinyl protein content was rather evenly distributed between the different membrane systems. In dormant embryos of barley and wheat acetyl CoA carboxylase was a soluble protein localized in the proplastids. During germination the biotin protein(s) became associated with the lamellar membrane fraction.