The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Further studies of the relationship between cation-induced chlorophyll fluorescence and thylakoid membrane stacking changes

Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b}$ light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.     

Effects of cations on the adhesion between membrane vesicles obtained by digitonin fractionation of spinach chloroplasts

The heavy fraction obtained by digitonin treatment of stacked spinach chloroplasts, suspended in media with different ionic composition, was examined by electron microscopy. In the presence of 5 mM MgCl₂ the thylakoid fragments adhere to one another in a ‘stacked configuration,’ while, in the presence of 10 mM NaCl, mainly only single ‘unstacked’ vesicles are present, which, upon addition of 5 mM MgCl₂, completely revert to the stacked configuration. As previously reported (Chow, W.S. and Barber, J. (1980) Biochim. Biophys. Acta 593, 149–157), no difference in fractionation of chlorophyll between light and heavy fractions was seen after a second digitonin treatment of this fraction suspended in media containing different cation concentrations. From these results it was concluded: (1) that for the unstacking process the movement of proteins or complexes from the stromal to the granal lamellae is not required. Upon lowering the screening by cations of the surface negative charges, the membranes separate from one another; (2) that, under these conditions, as in others (Jennings, R.C., Gerola, P.D., Garlaschi, F.M. and Forti, G. (1980) FEBS Lett. 115, 39–42), digitonin fractionation is not a tool to investigate the degree of membrane stacking.     

Morphological and Photosynthetic Properties of Digitonin-treated Chloroplast Membranes from the Wild-type and ac-5 Strains of Chlamydomonas reinhardi

Chloroplast membranes of wild-type Chlamydomonas reinhardi, treated with digitonin, yield photosystem II-rich and photosystem I-rich fractions; this fractionation is accompanied by a separation of stacked (grana) lamella from unstacked (stroma) lamellae. Poor fractionation of the photosystems occurs when the treated chloroplast membranes derive from the ac-5 strain grown mixotrophically, whereas good fractionation occurs with ac-5 cells grown phototrophically; the mixotrophic cells possess only unstacked membranes, whereas the phototrophic cells possess stacked membranes. We concluded that digitonin fractionation is dependent on the stacked membrane configuration.     

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.     

The effects of cations and trypsin on extraction of chlorophyll-protein complexes by octyl glucoside

The extraction of chlorophyll-protein (CP) complexes from thylakoids by the detergent octyl glucoside is strongly affected by pretreatment of the thylakoids with trypsin or cations. In these experiments, washed thylakoids were incubated in the presence of 0.5 μm to 5 mm Mg²⁺, pelleted, and extracted with octyl glucoside (30 mm). Increasing amounts of Mg²⁺ depressed extractability of all CP complexes, but especially the chlorophyll a + b-containing light-harvesting complex (LHC). This cation effect is observed with other cations which promote thylakoid stacking (5 mm Mn²⁺ or Ca²⁺, 50 mm Na⁺). However, the effect is not merely due to stacking, since low concentrations of Mg²⁺ (0.5 μmto 0.5 mm) have a marked effect on extractability but have no effect on light scattering (OD 550 nm), an indicator of stacking. Furthermore, trypsin treatment of thylakoids stacked with 5 mm Mg²⁺ caused a significant reversal of stacking, but had little effect on extractability. Trypsin treatment of unstacked membranes resulted in increased extractability of all CP complexes, but especially of the LHC. Cation-treated membranes are also significantly different from those “stacked” at pH 4.5. While the latter do show decreased extractability, there is no change in the chlorophyll $\text{a}\text{b}$ ratio of the extract, and the membranes cannot be “unstacked” with trypsin. We conclude that octyl glucoside extractability reflects the lateral interaction of CP complexes with each other and with other components in the same plane of the membrane. It is clear that divalent cations have several effects on thylakoid membranes, not all of which are due to their ability to promote stacking.     

Bizonoplast, a unique chloroplast in the epidermal cells of microphylls in the shade plant Selaginella erythropus (Selaginellaceae)

Study of the unique leaf anatomy and chloroplast structure in shade-adapted plants will aid our understanding of how plants use light efficiently in low light environments. Unusual chloroplasts in terms of size and thylakoid membrane stacking have been described previously in several deep-shade plants. In this study, a single giant cup-shaped chloroplast, termed a bizonoplast, was found in the abaxial epidermal cells of the dorsal microphylls and the adaxial epidermal cells of the ventral microphylls in the deep-shade spike moss Selaginella erythropus. Bizonoplasts are dimorphic in ultrastructure: the upper zone is occupied by numerous layers of 2-4 stacked thylakoid membranes while the lower zone contains both unstacked stromal thylakoids and thylakoid lamellae stacked in normal grana structure oriented in different directions. In contrast, other cell types in the microphylls contain chloroplasts with typical structure. This unique chloroplast has not been reported from any other species. The enlargement of epidermal cells into funnel-shaped, photosynthetic cells coupled with specific localization of a large bizonoplast in the lower part of the cells and differential modification in ultrastructure within the chloroplast may allow the plant to better adapt to low light. Further experiments are required to determine whether this shade-adapted organism derives any evolutionary or ecophysiological fitness from these unique chloroplasts.     

Surface charges on chloroplast membranes as studied by particle electrophoresis

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts.2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge.3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface.4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues.5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting $\text{chlorophyl}\text{a}\text{chlorophyll}\text{b}\text{pigment · protein complex}$.6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts.7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory.8. Calculations of the ζ-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500–2000 Ų.9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

Surface charges on chloroplast membranes as studied by particle electrophoresis.

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts. 2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge. 3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface. 4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues. 5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting chlorophyll a/chlorophyll b pigment . protein complex. 6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts. 7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory. 8. Calculations of the zeta-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500--2000 A2. 9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

The role of chlorophyll-protein complexes in the function and structure of chloroplast thylakoids

Summary The photosynthetic pigments of chloroplast thylakoid membranes are complexed with specific intrinsic polypeptides which are included in three supramolecular complexes, photosystem I complex, photosystem II complex and the light-harvesting complex. There is a marked lateral heterogeneity in the distribution of these complexes along the membrane with photosystem II complex and its associated light-harvesting complex being located mainly in the stacked membranes of the grana partitions, while photosystem I complex is found mainly in unstacked thylakoids together with ATP synthetase. In contrast, the intermediate electron transport complex, the cylochrome b-f complex, is rather uniformly distributed in these two membrane regions. The consequences of this lateral heterogeneity in the location of the thylakoid complexes are considered in relation to the function and structure of chloroplasts of higher plants.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.