Nathan Nelson

Localization of genes for coupling factor subunits on the spinach plastid chromosome

Summary1)Messenger RNA obtained from spinach cotyledons directs the synthesis of all five CF1 subunits in vitro in a rabbit reticulocyte translation system. The alpha, beta and epsilon subunit polypeptides were found as translation products from ptRNA and whole-cell poly A−-RNA. The gamma and delta subunits were synthesized from whole-cell poly A+-RNA as precursors of substantially greater molecular weight indicating that they originate in the nucleus and are imported into the chloroplast. High resolution electrophoresis, immunoprecipitation with antibodies against individual CF1 subunits (Nelson et al. 1980), and proteolytic peptide mapping were employed to identify the products.2)The genes for alpha, beta and epsilon subunits of CF1 were located by hybrid-selected translation with matrix-immobilized ptDNA fragments of known map position. The genes for all three CF1 subunit polypeptides are located in the large single-copy segment (cf. Herrmann et al. 1980b) of the circular ptDNA and each gene appears to be present once on the chromosome. The genes for the beta and epsilon subunits lie near each other in immediate vicinity to the structural gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase. The gene for the alpha subunit is separated by approximately 40 kbp from this gene cluster, and located near the gene for the 32 kd photosystem II polypeptide (Driesel et al. 1980).3)Restriction fragments of spinach ptDNA with CF1 subunit genes were cloned into pBR 322 and used to construct detailed maps.

Genes and transcripts for the P700 chlorophylla apoprotein and subunit 2 of the photosystem I reaction center complex from spinach thylakoid membranes

A photosystem I reaction center complex has been purified to homogeneity by a procedure involving partial solubilization of spinach thylakoid membranes, ion exchange chromatography and centrifugation in sucrose gradients. The complex contains 7 polypeptides: the P700 chlorophylla apoprotein with an apparent molecular weight of 67 kd, which at high resolution splits into two bands, and smaller polypeptides of 22 (subunit 2), 18.5, 18, 16, 12 and 10 kd.Stable transcripts for the P700 chlorophylla apojprotein and subunit 2 were found in plastid and cytosolic RNA, respectively. The apoprotein product obtained by translation in a mRNA-dependent cell-free rabbit reticulocyte lysate and also by DNA-programmed transcription-translation of cloned plastid DNA fragments inE. coli lysates was indistinguishable immunologically and electrophoretically from the authentic protein. However, the product immunologically related to subunit 2 was 4 kd larger than the mature compound indicating that this protein is encoded in the nucleus and synthesized as a precursor.The gene for the P700 chlorophylla apoprotein has been physically mapped on the spinach plastid chromosome by hybrid selection mapping and DNA-programmed cell-free transcription-translation using cloned restriction fragments of plastid DNA. There is one gene copy per chromosome and it is located centrally in the large single-copy region of the circular DNA molecule. This gene is uninterrupted and is transcribed in the same direction as that of the large subunit of ribulose bisphosphate carboxylase/oxygenase. Its transcript is approximately 4 kb longer than the 2 kbp structural gene.

Proton–ATPase of Chloroplasts

Publisher Summary This chapter discusses proton–ATPase of chloroplasts. It was demonstrated that upon reconstitution of the proton ATPase into phospholipid vesicles, the enzyme system can utilize an artificial proton gradient for ATP formation. In a study described in the chapter, proton–ATPases were identified and isolated from several energy transducing membranes. They are composed of two distinct structures: (1) a catalytic sector that is hydrophilic in nature and (2) a membrane sector that is hydrophobic in nature. The chloroplast proton–ATPase complex possesses similar structures and thus, they are applicable for the rest of the energy transducing membranes. Chemical reactions must be involved in the translation of proton motive force into the chemical energy in ATP. There are strong indications that for every electron transferred from water to a Hill acceptor, two protons are pumped inside the thylakoid membrane. The measured ΔpH contained sufficient energy to drive phosphorylation against its maximum potential, provided three protons are used by the ATPase complex per one ATP formed. Therefore, there is a possibility that the P: 2e ratio for noncyclic photophosphorylation is about 1.3. The mechanism by which three protons are involved in a single chemical event is difficult to visualize, and it might be that the third proton imposes a proper conformation in one of the catalytic steps. However, it is still unknown whether phosphate is covalently bound to the enzyme during the phosphorylation activity of proton ATPases

Genes and transcripts for the ATP synthase CF0 subunits I and II from spinach thylakoid membranes

SummarySubunits I and II of the ATP synthase CF0 in spinach are encoded by plastid and nuclear genes, respectively. Using hybrid selection translation, cell-free transcription-translation of cloned restriction fragments and an appropriate antiserum, a single gene for subunit I has been located on the circular plastid chromosome between the genes for the ATP synthase proteolipid and subunit alpha. This finding implies that the five plastome-coded ATP synthase subunits in spinach are arranged in two clusters which are separated by approximately 40 kilobase pairs of DNA. The genes of each cluster appear to be transcribed into large RNA species. The analysis indicates that the putative polycistronic proteolipid/CF0-I/alpha transcript is post-transcriptionally modified. The previous assumption that the structural gene for subunit I is split has not been excluded.-The transcript for subunit II has been found in the cytosolic RNA fraction. It lacks complementarity to plastid DNA and appears to be decoded into a protein precursor.-Evidence is presented that spinach chloroplast ATP synthase contains more than eight subunits.

Biogenesis of photosystem I reaction center during greening of oat, bean and spinach leaves.

SummaryThe relative amounts of some chloroplast polypeptides were followed during greening of leaves from three different plant families. Oat, bean and spinach were the representatives of the Gramineae, Leguminosae and Chenopodiaceae, respectively. By using specific antibodies against subunits of the chloroplast protein complexes, it was found with that method that the protein complexes which are not involved in a photobiochemical reaction were synthesized in etiolated leaves and their amounts did not significantly change during greening. Examples of these are the large and small subunits of ribulose 1,5-bisphosphate carboxylase, the subunits of the chloroplast coupling factor (CF1) and cytochrome b6-f complex. On the other hand, in photosystem I reaction center, the synthesis of subunits II, III, IV and V was found to be induced by light. Sequential synthesis of these subunits was observed. Subunit II is the first to be synthesized after exposing the plants to light. The synthesis of subunits III, IV and V followed the synthesis of subunit II in this order. Subunit I of photosystem I reaction center was present in etiolated leaves and its amount was not significantly altered during the first few hours of greening.

Photosystem I reaction centers fromChlamydomonas and higher plant chloroplasts

A photosystem I reaction center has been isolated fromChlamydomonas chloroplasts and compared with the photosystem I reaction center from higher plants. While the higher plant reaction center is active in cytochrome 552 photooxidation, theChlamydomonas preparation was not active unless salts were included in the assay medium or the pH was lowered to 5. Subunit III-depleted photosystem I reaction center from higher plants is also inactive in cytochrome 552 photooxidation in the absence of salts. As with theChlamydomonas reaction center, salts induced its activity. Subunit I of the photosystem I reaction center has tentatively been identified as the binding site of cytochrome 552.

Electron spin resonance studies of bound ferredoxin in chloroplast photosystem I reaction center

The primary photochemical reaction in chloroplast photosystem I has been identified as the photooxidation of a reaction center chlorophyll complex PToo [l] . Bound ferredoxin was proposed as the primary electron acceptor for this reaction [2,3]. However, recently an alternative primary electron acceptor was suggested based on the observation of a light induced EPR signal at g = 1.76 under conditions in which the bound ferredoxin was fully reduced [4--61. We have recently isolated a photosystem I reaction center which was active in NADP photoreduction and contained 5-6 polypeptides and PToo reaction center associated with a single polypeptide [7,8]. It is the purpose of this work to follow the bound ferredoxin content during the purification steps of P7eo reaction center.

Purification and composition of photosystem I reaction center of Prochloron sp., an oxygen-evolving prokaryote containing chlorophyll b

The photosystem I reaction center complex of the photosynthetic prokaryote Prochloron, present as a symbiont in ascidians from the Red Sea at Eilat (Israel), was isolated and characterized. The complex consists of 4 polypeptide subunits, and therefore is similar to that of cyanophytes and green algae. Their apparent molecular masses (in kDa) are about 70 (subunit I), 16 (subunit II), 10 (subunit III), and 8 (subunit IV). The purified reaction center contains about 40 chlorophyll molecules per P‐700 as compared to about 800 in the intact thylakoids. Subunit I has the same apparent electrophoretic mobility and appears as a double polypeptide band as reported for this subunit in higher plants and algae. Immunological cross‐reactivity was detected among subunits I and II of Prochloron and photosystem I reaction centers of higher plants and algae.

PROTON CHANNELS IN CHLOROPLAST MEMBRANES

The participation of proton-translocating ATPase in energy transducing membranes was postulated in the Mitchell hypothesis. Since then it was isolated from a wide variety of energy-transducing membranes, including membranes of mitochondria,? chloroplast^,^ chromatophors, and b a ~ t e r i a . ~ In all cases it is composed of a catalytic sector, which is hydrophylic in nature, and a membrane sector, which is hydrophobk6 The chloroplast proton-ATPase is composed of eight different polypeptides, five of which comprise the catalytic sector (CF,) and the other three, the membrane sector (CF,) of the complex. The function of CF, is to catalyze the phosphorylation of ADP to ATP, and the function of CF, is to transfer the protons from the internal side of the membrane to the catalytic part of the enzyme. The catalytic sector CF, can be partially removed from chloroplast membrane by EDTA treatment and it can be totally removed by sodium bromide t r e a t m e ~ ~ t . ~ . ~ ~ It was clearly demonstrated that upon removal of CF, the chloroplast membrane becomes leaky to protons and that this leak can be blocked by the addition of purified CF, or by the energy transfer inhibitor N,N1-dicyclohexylcarbodiimide (DCCD) . x v l1 The mechanism of the proton conduction across the chloroplast membrane is not fully understood. It is almost certain that subunit 111 of CF,, (the chloroplast proteolipid) plays the main role in the proton conduction and that this subunit binds the DCCD.. l2 It was shown that, upon reconstitution of the purified proteolipid into lipid vesicles, DCCD-sensitive proton conduction across the lipid membrane was created.. Whether this proton conduction occurs via channels, carriers, or some mixture of both is yet to be decided. Studies on the structure and function of CF, might shed light on the mechanism of proton conduction across membranes and might help to elucidate the mechanism of phosphorylation driven by proton gradients.

Properties of a novel ATPase enzyme in chromaffin granules

Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae. The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with [γ-32P]-ATP and decoration with [125I]concanavalin A and [125I]protein A with antibody that was raised against chromaffin-granule membranes. It was found that the membranes cross-contaminated each other by less than 10%. The technique of immunodecoration with antibodies against β subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, andE. coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes. It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes. The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes. The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan. It was much less sensitive than the mitochondrial membranes to antibody against β subunit of proton-ATPase fromE. coli membranes. After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated. The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.