A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

Inside-out vesicles derived from grana partition regions of spinach thylakoids were isolated by Yeda press treatment followed by aqueous polymer phase partitioning. Their structure was examined by freeze-etch electron microscopy, which revealed numerous tetrameric particles on their outer surface, corresponding to the ESs particles of the lumenal surface of intact thylakoids. The vesicles were treated with several different salt washes which specifically removed some or all of the extrinsic polypeptides of the oxygen evolving complex. Removal of the 16 and 23 kD extrinsic polypeptides with NaC1 caused a reduction in the surface relief or loss of the tetrameric structure of the ESs particles. When the 33 kD polypeptide was removed in addition to the 16 and 23 kD polypeptides, either by washing with CaCl: or alkaline Tris, the particles could no longer be clearly resolved from the membrane surface. Vesicles washed with 1 M-CaCl 2 were reconstituted with a 10-fold excess of a crude extract containing the extrinsic polypeptides of the oxygen evolving complex. This resulted in the reappearance of particles on the membrane surface, although they did not have a clear tetrameric structure. Thus the extrinsic polypeptides of the oxygen evolving complex are the major components of the tetrameric ESs particles of thylakoids. I N T R O D U C T I O NA characteristic feature of higher plant thylakoids is the presence of particles with a tetrameric structure of the inner surface of grana partition regions (2 l, 26). They are revealed by freeze-etching and are designated ESs particles using the terminology of STAEHELIN (25), and correspond to the quantosomes of PARK and BIGGINS (20). They are the surface projections of the membrane spanning EFs particles which, under certain circumstances (13, 14) form ordered arrays. The correspondence between EFs and ESs arrays enabled this correlation to be established (14).The composition of the EFs/ESs particle has been the subject of several papers. The first suggestion that they were the structural counterpart of photosystem II came from the digitonin fractionation studies of ARNTZEN et al. (7). This was substantiated by quantitative analysis of the Abbreviations: Chl = chlorophyll; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; EFs = endoplasmic fracture face of stacked thylakoids; ESs = endoplasmic surface of stacked thylakoids; LHCII = light-harvesting chlorophyll production of photosystem II; Mes = 2-(N-morpholino)ethanesulphonic acid; PFs = protoplasmic fracture face of stacked thylakoids; Tris = tris(hydroxymethyl)aminomethane.Springer-Verlag

Section: Resultsmentioning

confidence: 99%

Extrinsic polypeptides of the chloroplast oxygen evolving complex constitute the tetrameric ESs particles of higher plant thylakoids

Simpson

Andersson

1986

“…Grana membranes were isolated using a modification of the Triton X-100 method of BER-THOLD et al (7) as described by MOLLER and HoJ (22). The membranes were solubilized using dodecyl-~,D-maltoside and fractionated into water-soluble polypeptides derived from the oxygen-evolving site, LHCII, and the reaction center complex of photosystem II (RC) by centrifugation on 10-30% sucrose gradients containing 10 mM-Tricine, pH 8.0 and 0.025% Triton X-100 (9, 14).…”

Section: Isolation Of Chl-protein Complexesmentioning

confidence: 99%

The light-harvesting complex of photosystem II in barley. Structure and chlorophyll organization

Hinz

Welinder

1987

The isolated light-harvesting complex of photosystem II (LHCII) in barley had a chlorophyll (Chl) a/b ratio of 1.37 (mol/mol) and contained an average of 13.1 Chl molecules per 25 kD polypeptide chain, corresponding to 7.6 molecules of Chl a and 5.5 molecules of Chl b. Tryptic digestion of native LHCII yielded a 16 kD fragment which contained 12 of these 13 Chl molecules. The 16 kD fragment started with the serine 52 of the mature polypeptide, as determined by N-terminal amino acid sequence analysis and comparison with the amino acid sequence deduced from the nucleotide sequence of a corresponding gene in wheat (LAMPPA et al. 1985). Circular dichroism measurements of LHCII in 1% SDS or at extremes ofpH, showed that there were at least two populations ofChl a and that Chl a was much more sensitive to changes in the aqueous environment than Chl b. We present a model for the folding of LHCII with four transmembrane helices, based on the above results and on the application of the rules of CHou and FASMAN ( 1978 ) and PAUL and ROSENBUSCH ( 1985) for the prediction of secondary structure using the wheat sequence.

“…Taken together these results imply that this protein is not composed of subunits. The protein as judged by chromatofocusing is relatively acidic eluting at pH 4.6 ( Figure 3), and therefore probably has a pI slightly higher than 4.6 (26). During the purification of malonyl-CoA:ACP transacylase from whole leaf homogenates, the activity profiles were single headed and the elution positions of enzyme activity upon gel filtration, Blue Sepharose chromatography, ion exchange and chromatofocusing were identical to those obtained when using chloroplast stroma proteins as starting material.…”

Section: Purification and Characteristics Of Malonylcoa:acp Transacylasementioning

confidence: 73%

Barley acyl carrier protein: Its amino acid sequence and assay using purified malonyl-CoA:ACP transacylase

Høj

Svendsen

1983

Self Cite

Keywords: Fatty acid synthetase, c y a n o g e n b r o m i d e , c a r b o x y p e p t i d a s e Y, c h r o m a t o f o c u s i n g , s t r u c t u r e -f u n c t i o n relationship.MalonyI-CoA:ACP transacylase from barley (Hordeum vulgare L.) has been purified to homogeneity and used in an assay for acyl carrier protein (ACP). The transacylase is an acidic, monomeric protein with a molecular weight of 34,500 very similar to the analogous E. coli enzyme. A heat and acid stable acyl carrier protein from barley has been purified to homogeneity and its chemical composition determined. The ACP consists of a continuous stretch of the following 72 amino acids H2N-A-A-M-G-E-A-Q-A-K-K-E-T-V-D-K-V-(C?)-M-•-V-K-K-Q-L-A-V-P-D-G-T-P-V-T-A-E-S-K-F-S-E-L-G-A-D-S-L-D-T-V-A comparison of the primary structure of this plant ACP and bacterial ACP reveals two identical sequences (underlined) in the midregion of the molecule containing the 4'-phosphopantetheine attachment site, while differences occur outside this region. Nine extra residues (italicized) are present at the N-terminal end of the barley protein thereby accounting for its larger size. Identical products are obtained when barley chloroplast fatty acid synthetase is incubated with either barley or E. coli ACP, but the latter is twice as active as the former in fatty acid synthesis. The possible significance of the Nterminal part of the ACP is discussed in relation to the reported differences in biochemical activities of plant and bacterial ACPs.Abbreviations: ACP = acyl carrier protein, BSA = bovine serum albumin, CoA = coenzyme A, DTT = 1,4-dithiotreitol, fas = fatty acid synthetase, Hepes = N-2-hydroxyetbylpiperazine-N'-2-ethanesulfonic acid, kD = kilodalton, Mes = 2-(N-morpholino)ethanesulfonic acid, Mops = 3-(N-morpholino)propanesulfonic acid, Pipes = piperazine-N,N'-bis(2-ethane sulfonic acid), PTH = phenylthiohydantoin, SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis, Tricine = N-(tris(hydroxymethyl) methyl) glycine, u.v. = ultra violet.Springer-Verlag