Gunilla Høyer-Hansen

The resolution of chlorophyll a/b binding proteins by a preparative method based on flat bed isoelectric focusing

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non‐ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.

Changes in the polypeptide composition of internal membranes of barley plastids during greening

A method is described for obtaining good yields of purified internal membranes of plastids from greening barley seedlings. The procedure is suitable, without modification, for isolating intact plastids from etiolated seedlings and all early stages of greening. Electron microscopy was used to identify the types of contaminants present in preparations obtained by different methods, and to assess the degree of contamination in purified membranes. Gel electrophoresis of the sodium dodecyl sulphate-solubilised membrane polypeptides established that the proteins pelleted during flotation were a major component of unpurified membrane preparations from etioplasts and plastids greened for 9 hours or less. The polypeptide composition of purified etioplast membranes consisted of 35 bands. At least 15 of these disappeared during subsequent greening. After only 3 hours of illumination, the internal membranes contained almost all of the 43 polypeptides present in mature chloroplast thylakoids. The changes in polypeptide composition from 3–24h were mainly quantitative in nature.

Chlorina mutants of barley (Hordeum vulgare L.)

We have examined 31 newchlorina mutants of barley using in vivo absorption spectroscopy, 77 K fluorescence emission spectroscopy, room temperature fluorescence induction kinetics, HPLC separation of pigments and SDS-PAGE. Based on these properties they can be placed into 4 groups. The first group consists of 10 mutants which are allelic to the chlorophyllb-lesschlorina-f2 and comprises five strongly and three slightly leaky mutants at this locus. The decrease in chlorophyllb content was correlated with a corresponding decrease in the amount of chlorophylla/b-proteins in the thylakoids. One mutant (chlorina 106) was found which had a very low chlorophyllb content and a deficiency in Chla/b-P2, but was not allelic tochlorina-f2. The second group ofchlorina, mutants has unusual fluorescence properties, with a high Fm/Fo ratio. Gaussian deconvolution of the 77 K fluorescence emission spectra revealed an increase in the amplitude of components emitting at 694 and 718 nm. The possibility is discussed that these properties result from the absence of the PSI connecting antenna LHCI-680. The third group has a Fs/Fo ratio <1, and other properties consistent with a partial deficiency in the chlorophylla-proteins of PSII reaction centres. The fourth group consists ofchlorina mutants which are very similar to wild-type in the properties examined, differing mainly in having a lower chlorophyll content.

Identification of haem-proteins in thylakoid polypeptide patterns of barley.

Thylakoid polypeptides from barley were separated by polyacrylamide gel electrophoresis by use of either SDS or LiDS as the detergent. Staining of either gel-type with 3,3′,5,5′-tetramethylbenzidine-H2O2 revealed two barley polypeptides with peroxidase activity. The same two polypeptides were shown to incorporate [14C]-δ-aminolaevulinic acid, identifying haem as their prosthetic group. The haem-protein with a molecular weight of 33,000 is cytochromef and that with a molecular weight of 20,000 is suggested to be a subunit of cytochromeb6. The two polypeptides are also present in etiplast membranes, which from prior spectroscopic evidence are known to contain cytochromesf andb6.

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløfs Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb63) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k23, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.

Freeze-fracture evidence for the isolation of inside-out spinach thylakoid vesicles

An aqueous dextran/polyethylene glycol two phase system is used to separate spinach chloroplast thylakoid vesicles obtained by Yeda press treatment of a grana-enriched fraction. It is shown by freeze-fracturing and freezeetching that the majority of the vesicles in the dextran-rich bottom phase are turned inside-out with respect to the surfaces of the thylakoid in the chloroplast. Most of the vesicles in the top phase had the normal orientation of the two thylakoid surfaces. These results confirm the previous proposal that the bottom phase vesicles show light induced proton extrusion instead of uptake because their membranes are turned inside-out. The polypeptide pattern of the bottom phase thylakoid fraction is enriched in the light harvesting chlorophyll a/b protein complex and the reaction centre protein of photosystem II, which is consistent with the origin of these thylakoids from granal regions enriched in photosystem II. A mechanism is suggested to explain the formation of inside-out vesicles from grana.

CHARACTERIZATION OF SiX PUTATIVE PHOTOSYSTEM I MUTANTS IN BARLEY

Six putative photosystem I mutants in five different nuclear genes of barley have been characterized with respect to their photosynthetic electron transport capabilities,P700, cytochromes, chlorophyll-proteins and other thylakoid polypeptides. Three of these mutants (viridis-n34,−zb63, and−h15) have normal photosystem II activities but are wholly or partly deficient in photosystem I electron transport,P700, chlorophyll,a-protein 1 and its apoprotein. These three mutants also lack low molecular weight polypeptides which are believed to be integral photosystem I iron-sulfur centres. Of the remaining mutants,xantha-q75 and−q76, which are allelic, are mainly depleted in photosystem II and the other (viridis-q42) had aout 75% of the wild-type activity in both photosystems. The data are discussed in relation to the fluorescence induction kinetics and low temperature fluorescence emission spectra of the mutants.

A cDNA clone encoding a 10.8 kDa photosystem I polypeptide of barley

A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS‐polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15 457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane‐spanning regions.

Visualization of antibody binding to the photosynthetic membrane: the transmembrane orientation of cytochrome b-559.

We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.

Photosystem II and cytochromeb-559 in the stroma lamellae of barley chloroplasts

The presence of photosystem II polypeptides and photosystem II activity in unstacked regions of barley thylakoid membranes were investigated by immunological and spectroscopic techniques. Immunogold labelling of Lowicryl-embedded chloroplasts showed that PSI and the ATPase were completely excluded from stacked regions, while a significant proportion of PSII was located in unstacked regions of the membrane. A membrane fraction derived from stroma, lamellae was shown by immuno-blotting to contain PSII components. Their abundance was approximately one tenth (on a chlorophyll basis) of that in the starting material. The stroma lamellae showed a corresponding level of photosystem II-mediated electron transport, in the presence of the electron donor diphenyl-carbazide. Much of the activity was DCMU insensitive, and the reaction centres lacked the antennae pigment CP29, which was located only in appressed thylakoids. Cytochromeb-559 LP, but not HP, was present in the stroma lamellae fraction. Evidence is presented that the native low potential form of cytochromeb-559 exists as a unique molecular species, distinct from the high potential form (cytochromeb-559 HP).