Investigations of the role of the main light-harvesting chlorophyll-protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex.

The functions of the light-harvesting complex of photosystem II (LHC-II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication-freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.

Freeze-fracture analysis of membrane appression and protein segregation in model membranes containing the chlorophyll-protein complexes from chloroplasts.

Cation-induced membrane appression and lateral segregation of chlorophyll-protein complexes have been investigated by freeze-fracture analysis of model membranes containing photosystem 1 and the light-harvesting complex of photosystem 2. In light-harvesting complex proteoliposomes, cations caused extensive membrane adhesion and a segregation of protein into appressed regions. A marked flattening of the appressed membranes, sometimes together with a co-alignment of the particles on the opposing membrane faces, strongly suggests a direct transmembrane attraction between the protein particles. Photosystem 1 membranes were not appressed by cations but some clustering of the particles occurred, together with their exclusion from some regions of the lipid. By incorporating dipalmitoylglycerophosphocholine into the membranes, it is shown that a similar exclusion of the particles can occur due to liquid-crystalline to gel state transitions of the lipids. Proteoliposomes containing both the light-harvesting complex and photosystem 1 displayed cation-induced membrane appression, but only between regions containing the light-harvesting complex. Photosystem 1 was largely confined to unappressed membranes. Destacking occurred at low salt concentrations irrespective of whether photosystem 1 was present, showing that in proteoliposomes, and probably in thylakoids, this process does not require the presence of highly charged complexes from unappressed regions.

Immunological evidence for apoproteins of the light-harvesting chlorophyll-protein complex in a mutant of barley lacking chlorophyll b.

A barley mutant lacking chlorophyll b and the pigmented light-harvesting chlorophyll-protein of photo-system 2 is shown by several criteria to contain functional apoproteins of the light-harvesting complex. 1. Electrophoretic comparison of thylakoid polypeptide patterns, and the effects of trypsin treatment on these, suggests that the mutant contains several polypeptides equivalent in mobility to those of the wild-type complex. 2. An antibody monospecific for the light-harvesting complex agglutinated both wild-type and mutant thylakoids. 3. 'Western blot' immunoelectrophoretic analysis indicated that of four distinct subunits of the light-harvesting complex in the wild-type thylakoids, three are detectable in the mutant. 4. As in wild-type lamellae at least one of the light-harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light-harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.

Transbilayer organization of the chlorophyll-proteins of spinach thylakoids.

To investigate the transverse bilayer organization of the chlorophyll-proteins of the three intrinsic chlorophyll-protein complexes, the effects of proteolytic enzymes, and an antibody against the light-harvesting complex were compared using right-side-out and inside-out thylakoid vesicles. The vesicles were isolated by aqueous polymer phase partitioning following the fragmentation of spinach thylakoids by passage through a Yeda press. Both vesicle types were agglutinated by an antiserum specific for the light-harvesting complex, although proteolytic degradation of the complex occurred only in right-side-out vesicles. In addition, there are different antigenic sites for the light-harvesting complex on the inner and outer thylakoid surfaces. Polypeptides of the chlorophyll-alpha-protein complex of photosystem II were degraded by proteases at both membrane surfaces. We concluded that both these chlorophyll-protein complexes are membrane spanning and transversely asymmetric, but that the light-harvesting complex polypeptides accessible at the inner thylakoid surface are more resistant to proteolytic attack. In contrast, the main chlorophyll-containing polypeptide (Mr = 64 500) of photosystem I complex was resistant to proteolytic attack at both the outer and inner thylakoid surfaces.

The role of the light-harvesting chlorophyll a/b protein complex in chloroplast membrane stacking. Cation-induced aggregation of reconstituted proteoliposomes.

The major intrinsic protein from spinach chloroplast membranes, the light-harvesting chlorophyll a/b-protein complex, contains two distinct polypeptides of Mr 23,500 and 26,000 and 31% lipid by weight, comprising five diacyl lipids and seven chlorophylls, together with some carotenoids, per 26,000-Mr polypeptide. The chlorophyll a/b ratio is 1.1. Low-temperature fluorescence emission spectra of the light-harvesting complex revealed a major peak at 681 nm with a shoulder of variable intensity at 695 nm. The 695-nm emission has been correlated with a progressive aggregation of the complex into two-dimensional, semi-crystalline sheets. To determine the role of the light-harvesting complex in cation-dependent thylakoid stacking, the purified complex has been quantitatively incorporated into liposomes containing the four major chloroplast diacyl lipids using a simple freeze-thaw technique. The proteoliposomes appeared largely as unilamellar vesicles, with diameters between 0.1 and 0.8 micron. Freeze-fracture analysis showed intramembrane particles of 8-10 nm corresponding to the incorporated complex. Both monovalent and divalent cations caused an immediate aggregation of the proteoliposomes, which was reversed at low cation concentrations and was largely inhibited by prior trypsin treatment. Since lipid vesicles themselves showed none of these effects, it is concluded that surface-exposed polypeptide regions of the light-harvesting complex are directly involved in thylakoid stacking in vivo.

The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant.

1. The subunit compositions of the F1 (oligomycin-insensitive) and F1--F0 (oligomycin-sensitive) mitochondrial ATPase complexes from Saccharomyces cerevisiae have been examined by the highly resolving technique of sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis using a discontinuous buffer system. When isolated in the presence of protease inhibitors, F1 and F1--F0 contained five and twelve bands, respectively; this contrasts with the four- and ten-band patterns seen previously using the less resolving disc gel method. When isolated in the absence of protease inhibitors both F1 and F1--F0 contain spurious polypeptides produced by proteolytic modification. 2. Endogenous protein turnover in S. cerevisiae was impaired in the presence of protease inhibitors. F1--F0 isolated from cells grown in the presence and absence of inhibitors contained an identical polypeptide composition, suggesting that the subunits are not significantly modified by endogenous proteases prior to cell harvesting. 3. Yeast F1--F0 prepared in the presence of protease inhibitors contains a latent, sodium dodecyl sulphate-activated protease contaminant. Sodium dodecyl sulphate-induced proteolysis is largely confined to the 52 000 dalton alpha subunit which degrades into polypeptides of 40 000 and 10 700 daltons. The 40 000 dalton band is apparently equivalent to the polypeptide previously designated subunit 3. 4. Both F1 and F1--F0 were isolated from Torulopsis glabrata, a yeast with considerably shorter mitochondrial DNA than that in S. cerevisiae. F1--F0 catalysed high rates of ATP--32Pi exchange when reconstituted into phospholipid vesicles, thus demonstrating the presence of a complete coupling mechanism. F1--F0 contained approximately twelve subunits and F1 five, like the S. cerevisiae complexes. It therefore appears that the shorter mitochondrial DNA length does not produce a significantly simpler ATPase subunit structure.

Studies of the oligomycin-sensitive ATPase from yeast mitochondria. Reconstitution of ATP-32Pi exchange in the presence of phospholipids.

A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin- and uncoupler-senstitive ATP-32Pi exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase-phospholipid-cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 A were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex.

Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide-adenine dinucleotide in barley.

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.