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

Steinback, Katherine E.; Burke, John J.; Arntzen, Charles J.

doi:10.1016/0003-9861(79)90381-3

Cited by 139 publications

(53 citation statements)

References 35 publications

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“…These results parallel observations that the LHCP of PSII is surface-exposed, since a 2,000-D segment could be removed by proteolysis (10,26 In the present study, we have also investigated the topography of PSI constituent polypeptides by pronase treatment of intact thylakoids followed by isolation of a modified PSI complex (25). This procedure differs from that previously used by Carter and Staehelin (10) …”

supporting

confidence: 85%

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

Ortiz¹,

Lam²,

Chollar³

et al. 1985

Plant Physiol.

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The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of -62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by I'4CFTNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by I"4CITNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronasetreated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed. complexes to the overall structure of the chloroplast membrane is less well understood.It has recently been possible to isolate all the chloroplast membrane electron transfer complexes in highly resolved functional form (7,16,25), and subsequently it has been demonstrated that these complexes are reconstitutively active in catalyzing noncyclic electron transport from water to NADP when supplemented with soluble protein cofactors (19). The availability of these resolved complexes allows for detailed examination of structure-function relationships in the individual complexes and examination of the nature of the interactions between complexes. In the present study, we have considered the topographical orientation of the chloroplast PSI complex. The impermeant chemical probe, TNBS,3 labeled with "1C, has been used to modify stroma-exposed amino acid side chains on proteins in unfractionated thylakoid membranes and reactive groups in the resolved PSI complex of Mullet et al. (25). The labeling pattern of PSI polypeptides in the membranes has been compared with that of the isolated PSI complex and subfractions isolated from this complex. Since any one sp...

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supporting

confidence: 85%

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

Ortiz¹,

Lam²,

Chollar³

et al. 1985

Plant Physiol.

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“…There were no significant differences in cation-induced stacking between RNH and RH thylakoids (data not shown). Both sets of membranes were maximally stacked at 150 mm NaCl as has been observed in both pea thylakoids (25) and in phosphatidyl choline vehicles containing pea LHC (23). Thus it appears that stacking characteristics could not account for differences in octyl glucoside extractability of RNH and RH thylakoids.…”

Section: Resultsmentioning

confidence: 73%

“…Vol. 81,1986 of thylakoid membranes have been correlated with the degree of stacking of those membranes (7,25). We studied cation-induced thylakoid stacking to determine whether changes in stacking could account for differences in octyl glucoside extraction of RNH and RH thylakoid membranes.…”

Section: Resultsmentioning

confidence: 99%

“…The washed thylakoids were resuspended in 10% (w/ v) glycerol, 65 mM Tris-HCl (pH 6.8), and 5 mM DTT, at a concentration of 1 mg Chl/ml, and stored at -7O°C (6). For cation treatments, thylakoids were thawed, washed in low salt medium (0.1 M sorbitol, 10 mm Tricine-NaOH [pH 7.8], 10 mM NaCi), and resuspended in a minimal volume oflow salt medium (25). An SDS.…”

Section: Methodsmentioning

confidence: 99%

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Low Temperature Development of Winter Rye Leaves Alters the Detergent Solubilization of Thylakoid Membranes

Griffith¹,

Hüner²,

Hayden³

1986

Plant Physiol.

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Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the 32Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.The ability of winter rye plants to photosynthesize efficiently at low temperatures is dependent on plant growth and development at low temperature (20). When Puma rye is grown at temperatures near freezing (2-5°C), leaves develop which are morphologically, anatomically and functionally distinct from leaves produced by plants grown at warmer temperatures (20C). These low temperature leaves have shorter blades, fewer stomates, thickened epidermal cell walls, increased epicuticular wax deposits and larger, multivacuolate mesophyll cells (9,12,17,18 5C (11, 18). No changes have been observed in Chl a/b, protein/Chl, Chl/P700 or PSII photosynthetic unit size (8,10,11,18) between the two membranes.In the absence ofdramatic changes in membrane composition, it was hypothesized that structural modifications of thylakoid membranes might account for the increased electron transport capacity which arises during the development oflow temperature leaves. Ultrastructural analyses of chloroplasts isolated from low temperature leaves show that they have the same number of granal stacks, although the grana tend to be smaller in size, than those of chloroplasts developed at warmer temperatures (18). Both sets of plastids exhibit similar particle densities on the exoplasmic and protoplasmic freeze-fracture faces of the thylakoids; however, the particle size distributions are significantly different on both fracture faces (18). Other modifications of rye membranes were revealed by SDS-PAGE of Chl-protein complexes extracted from thylakoids (8). For example, when 5...

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“…Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was carried out with a 10-17.5% linear polyacrylamide gradient slab gel with a 1-cm 5% stacking gel incorporating the buffer system of Laemmli (25). Samples were prepared for electrophoresis and gels were run, fixed, and stained for protein as described (26). Fluorography Chloroplast DNA Isolation and Restriction Endonuclease Analysis.…”

mentioning

confidence: 99%

Identification of the triazine receptor protein as a chloroplast gene product

Steinback

McIntosh

Bogorad

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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161

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The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem H. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed "B." The polypeptide that is believed to be a component of the B complex has recently been identified as a 32-to 34-kllodalton polypeptide by using a photoaffinity labeling probe, azido-[14Clatrazine. A 34-kilodalton polypeptide ofpea chloroplasts rapidly incorporates [3S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. roplasts results in-identical, sequential alterations of the 34-kldodalton polypeptide to species of 32, then 18 and 16 kldodaltons. From the identical pattern ofsusceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product ofchloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein ofphotosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. When dark-grown seedlings are transferred to light, a sequence of developmental processes leads to the formation of green, photosynthetically competent chloroplasts (1). Rapid accumulation of a major thylakoid protein of 32 kilodaltons (kDal) parallels the appearance offunctional activity (2). In Zea mays, this 32-kDal protein is structurally related to, and is presumably derived from, a 34.5-kDal polypeptide that is the major membrane-bound product of protein synthesis by isolated chloroplasts (3). It has been shown to be encoded by a chloroplast gene in Z. mays (4). A number ofchloroplast-encoded proteins appear to be synthesized in etioplasts of dark-grown maize seedlings (2), but the transcription of the chloroplast gene coding for this polypeptide is light dependent in developing plastids and has thus been described as a "photogene" (5, 6). Until recently, no function had been determined for the photogene 32 product; it has been identified now as a determinant of the electron transport function of a bound plastoquinone molecule in the photosystem II (PS II) complex (7). Its photoregulated synthesis (2, 8), apparent rapid turnover, and continued synthesis throughout all stages ofleafdevelopment (2, 3, 9) suggest a regulatory role for the protein product in PS II function. Purified PS II complexes isolated by detergent fractionation techniques contain a protein component of 32 kDal (10, 11). Certain mutants ofZ. nays lacking PSII function are deficient in a 32-kDal membrane polypeptide (12). Trypsin treatment of thylakoid membranes (13) or of isolated PS II complexes (10) results in the degradation of the 32-kDal polypeptide with concomitant loss of PS II activity. These lines of evidence also suggest that the 32-kDal polypeptide plays an integral role in PS II electron transport.A broad range of inhibitors of photo...

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

Cited by 139 publications

References 35 publications

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

Low Temperature Development of Winter Rye Leaves Alters the Detergent Solubilization of Thylakoid Membranes

Identification of the triazine receptor protein as a chloroplast gene product

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