The precursor of the major light-harvesting chlorophylla/b-proteins of photosystem II was synthesizedin vitro from a gene fromLemna gibba. When the labelled precursor was incubated with developing barley plastids, the precursor and the processed polypeptide were incorporated in the thylakoids in proportions that varied depending on the developmental stage of plastids. At early stages of development most of the precursor associated with the thylakoids could be removed by washing with 0.1 M NaOH, while in more mature plastids most of its was resistant to a NaOH wash. Insertion of the precursor into thylakoids required the presence of a stromal factor and Mg-ATP. The stromal factor is probably a protein. The insertion reaction has an optimal temperature of 25°C and a pH of 8. The appearance of the stromal factor and the thylakoid membrane's receptivity for the insertion of the precursor depended on the stage of plastid development. These observations are consistent with the hypothesis that the insertion of the precursor into the thylakoid prior to its proteolytic processing, is one of the steps involved in the assembly of the light-harvesting complex of photosystem II.

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A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.

MATERIALS AND METHODSTobacco (Nicotiana tabacum L. cv Turkish Samsun), spinach (Spinacia oleracea L. cv Northland), and pea (Pisum sativum L. cv Feltham First) were grown from seed in a soil-vermiculite mixture in a glass house. Thylakoid membrane preparation from leaves and assay of total thylakoid protein kinase activity were as previously described (18, 19), except that the amount of labeled ATP in the assay was increased to 10 uCi.Assessment of specific phosphorylation of the LHCP apoproteins was accomplished by halting the kinase assay by adding a final concentration of 3% (w/v) SDS. The samples, each containing 6 ,ug of Chl, were then electrophoretically fractionated on a 10% (w/v) acrylamide gel (15). Following electrophoresis, the gels were dried and the LHCP apoproteins, two closely migrating phosphoproteins of Mr approximately 28,000, were located by autoradiography. The 32P-labeled LHCP bands were excised and the amount of phosphate incorporated into them was measured from Cerenkov radiation in a scintillation counter. Assessment of phosphorylation of individual thylakoid polypeptides was carried out using the assay conditions described above, except that the Chl concentration in each 0.1 ml incubation volume was increased to 50 ,ug and each contained 200 gCi of [j-32p] ATP. Incubations were terminated after 3 min with 3% (w/v) SDS, and duplicate samples were electrophoretically fractionated on gels containing 7.5 or 12.5% (w/v) acrylamide and processed as above. Use of these two acrylamide concentrations facilitated fractionation of high and low mol wt polypeptides, respectively.

The light-harvesting complex (LHC) of photosystem 11 is composed of several different pigment-binding apoproteins. We have identified a cDNA clone LHCIIa-1 encoding the 31-kilodalton LHC Ila (CP29, Chi a/b-PI) apoprotein of barley (Hordeum vulgare). Direct protein microsequencing of an intemal peptide fragment from the LHC Ila apoprotein has been used to identify unequivocally the cDNA clone as that coding for the LHC Ila apoprotein. Microsequencing of the 28-kilodalton LHC lic protein (CP26) showed only minor sequence similarity to the LHC Ila protein, indicating that they are two different gene products. LHCIIa-1 codes for a protein of 286 amino acid residues (molecular weight, 31,308), which displays strong similarities to other pigment-binding LHC proteins, and yet contains an additional 42 amino acid residue segment. Two regions of strong intramolecular sequence similarity are also observed. The light-harvesting antenna complexes of PSI and PSII play an important role in the capture of light and its conversion into chemical energy. The LHCs2 surround each core complex and bind 50 to 80% of the Chl of a photosystem, thereby increasing the light-capturing ability of either photo-system. With refined native electrophoretic techniques used to separate detergent-solubilized thylakoid extracts, an increasing number of different LHCs associated with either PSI or PSII have been characterized. At present five LHC II and three LHC I pigment-protein complexes have been identified (3, 35), each with distinct biochemical characteristics. It isnot clear how each of the individual LHCs are arranged spatially with respect to the two core complexes. LHC Iha (CP29, Chl a/b-Pl) contains a single apoprotein of approximately 31 kD in barley (31). It is a relatively minor LHC II pigment-protein, binding approximately 3% of the total Chl (compared with approximately 45% for LHC Ilb) with a Chl a/b ratio of 2.25 to 2.50 (2, 31). protein has a red maximum at about 677 nm with a second somewhat unusual peak at approximately 643 nm that is characteristic of this complex (24, 31). Oxygen-evolving PSII core complex preparations have been isolated that contain LHC hIa but lack any of the other four LHC II components. It isthus inferred that LHC Ila is more tightly associated with the core, and the remaining LHC II complexes are more peripheral and/or less tightly bound to it (7, 15). All identified LHC apoproteins are coded for in the nuclear genome and are posttranslationally imported into the chlo-roplast, where they are inserted into the thylakoid membrane and bind pigments. Nucleotide sequences of cloned genes putatively coding for several different LHC I and LHC II proteins indicate that there is a high degree of sequence and probably structural relatedness among all of the LHCs (17, 33, 34). It has been postulated that LHC IIb, the major LHC II component, has a polypeptide(s) that contains three trans-membrane helices (4, 19; cf 21) with its amino terminus located in the stroma (29) and the carboxy terminus in the thylakoid lumen...