Alma Gal

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b 6/f‐less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light‐dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III‐S at comparable rates. However, the redox‐controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b 6/f‐less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b 6/f complex inhibited both the electron flow and duroquinol‐activated LHCII phosphorylation, but had no effect on the duroquinol‐dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox‐activated protein kinase(s). A quinone‐binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b 6/f complex.

The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen-located polyphenol oxidase

Phosphorylation of chlorophyll a/b‐binding proteins of the of photosystem II light‐harvesting assembly controls the energy distribution between the two photosystems as well as the turnover of thylakoid membrane proteins. The LHCII kinase, suggested to be a 64 kDa protein, is light‐regulated by a mechanism involving reduction of plastoquinone and the participation of the cytochrome b 6/f complex. A cDNA encoding that protein has been isolated from a lambda gt11‐based library made from spinach polyadenylated RNA using a two‐step strategy involving screening by polyclonal monospecific antisera and plaque hybridization. The protein of 73.1 kDa molecular mass represents a precursor which contains a bipartite transit peptide of 101 amino acid residues (11.0 kDa) that directs the protein into the thylakoid lumen. It can be phosphorylated in vitro, and exhibits significant homology to plant polyphenol oxidases, not to kinases. The gene was therefore designated PpoA. Reinvestigation of components in the molecular mass range of 50–70 kDa disclosed five additional proteins which can accompany kinase‐active cytochrome b 6/f, photosystem II and AMS [1] preparations. Four of them can be phosphorylated in vitro; two with apparent molecular masses of 53 and 66 kDa are capable of phosphorylation, and represent new, yet unidentified proteins.

Phosphorylation of cytochrome b6 by the LHC II kinase associated with the cytochrome complex.

The cytochrome b6 polypeptide present in cytochrome b6/| preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox‐controlled LHCII kinase. The N‐terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

Characterization of light-activated reversible phosphorylation of a chlorophyll a/b antenna apoprotein in the photosynthetic prokaryote Prochlorothrix hollandica

Abstract Phosphorylation of thylakoid proteins of 35.25.23 and 14 kD was shown to occur in Prochlorothrix hollandica both in vivo and in vitro. In vivo: the kinase was active in high light, but not in low light or dark. Next to the 35 kDa protein, which formed the major target, 25 and 23 kDa proteins were phosphorylated. PS II light (650 nm) stimulated kinase activity, which was inactive in PS I light (710 nm). In vitro: the kinase was found to be constantly active, and inhibitors of photosynthetic electron transport had no effect on phosphorylation levels. Prolonged dark incubation before isolation of thylakoid membranes did yield a partially inactivated kinase. Rapid activation could be obtained by both light and addition of the plastoquinol analogue duroquinol. Phosphorylation of the 23 and 25 kDa proteins was enhanced by addition of antimycin A under illuminating conditions and by sodium fluoride. The latter observation suggests that thylakoid proteins may be phosphorylated by the same kinase, but are dephosphorylated at different rates. The 35 kDa phosphoprotein was shown to form a chlorophyll a/b antenna copurifying with PS I. Light-regulated reversible phosphorylation of a chlorophyll a/b binding protein in P. hollandica immunologically related to that of Prochloron indicates that a similar mechanism may operate in the latter. The hypothesis was raised that reversible phosphorylation of a 35 kDa apoprotein of the chlorophyll a/b antenna results in a change in its orientation to PS I by which it enables/enhances energy transfer to PS II. It is suggested that this antenna is structurally associated with PS I. Lastly, a 33 kDa polypeptide was identified as a chlorophyll a/b binding protein which may form an antenna to PS II.

Reconstitution of the Turkey erythrocyte adenylate cyclase sensitivity to l-epinephrine upon re-insertion of the Lubrol solubilized components into phospholipid vesicles

Abstract Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (−)epinephrine. The 125I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution.

Photoinactivation of photosystem II and degradation of the D 1 protein are reduced in a cytochrome b6/f-less mutant of Chlamydomonas reinhardtii.

Abstract The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB charge recombination, rise in in trinsic fluorescence and loss of variable fluorescence. These parameters relate to functions in volving the reaction center II D1 protein. The light-induced degradation of D1 in the mutant cells was also considerably reduced, with a t 1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.

The in vitro reconstitution of rough endoplasmic reticulum membrane derived from human placenta

Abstract The isolation of rough endoplasmic reticulum from human placenta is described. Puromycin facilitated the detachment of the majority of the ribosomes from the membrane. Ribosomes could be re-attached to the stripped membrane, and puromycin was also necessary for detaching the rebound ribosomes, indicating that peptidyl-tRNA anchors the ribosomes to the membrane in both the native and the reconstituted rough membrane. Peptides labeled in-vitro on membrane bound ribosomes (native and reconstituted rough membrane) remained associated with the membrane after the ribosomes were detached. A protein with an electrophoretic mobility in SDS gels identical to that of HPL is among the membrane associated nascent proteins of the native and the reconstituted rough membrane.

AFFINITY LABELING BY ATP AND GTP OF THE NUCLEAR ENCODED OEC23 PROTEIN OF THE PHOTOSYSTEM II OXYGEN EVOLVING COMPLEX

Abstract The isolated OEC23 (oxygen evolving complex) protein of Photosystem II can be affinity-labeled by ATP and GTP in the presence of UV light. Labeling is competed by ATP, AMP, and NAD as well as by the ATP and ATP-γ-S and 5′-p-Fluorosulfonylbenzoyl-adenosine (FSBA) and is inhibited by Dithiothreitol (DTT). The physiological significance of this finding and the possible implication of ATP and GTP binding in thylakoid membrane dynamics remains to be further investigated.

LHCII Kinase Activation During Greening Correlates with the Assembly of Cytochrome b6/f Complex and Grana Formation

LHCII kinase activity was reported to be regulated by its association with the cytochrome b6/f complex in vivo and in vitro in mature plants and green algae 1-5. The goal of this work was to find out whether this relationship exists also during the greening process and thylakoid membrane formation. The assembly of the cytochrome b6/f complex is assumed to be a gradual process consisting of synthesis of its subunits both in the cytoplasm (Rieske Fe-S) and in the chloroplast (cyt f, b6, subunit IV) and their proper insertion in the thylakoid membrane. The natural substrates, LHCII polypeptides, are also synthesized, processed and inserted into the membrane in a gradual process concomitant with the greening and formation of the grana stacks. However there was no available information regarding the appearance of LHCII kinase during the greening. Previous data indicated that LHCII phosphorylation during the greening of Chlamydomonas reinhardtii y-1 mutant occurs 5–8 hours after the onset of the greening 6. This is also the time at which grana stacks containing more than 2 paired thylakoids can be observed. Thus it was of interest to find out whether the rise in LHCII kinase activity was due to the gradual appearance of the enzyme or to the functional assembly of the cytochrome b6/f complex accompanied by the structural organization of the grana stacks. The results presented in this work, based on greening systems in Chlamydomonas y-1 mutant as well as of dark grown spinach, support the concept that cytochrome b6/f mediates the LHCII kinase activation and indicate that the assembly of the cytochrome complex is related to the activation of the pre-existing enzyme to phosphorylate the LHCII polypeptides in a redox dependent manner.

Specific Loss of LHCII Phosphorylation in a Chlamydomonas Mutant Lacking the Cytochrome b6/f Complex

Recent studies in our laboratory have indicated that the cytochrome b6/f (cyt.b6/f) complex is involved in the light induced phosphorylation of LHCII polypeptides in Acetabularia and Lemna (1,2). Similar data have been obtained with several cyt.b6/f deficient Chlamydomonas (3) and maize mutants (4,5). To further ascertain that indeed the cyt.b6/f complex is required for the activation of the redox controlled LHC II kinase, the activity of this enzyme(s) was assayed in another Chlamydomonas mutant lacking the complex components.