The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination

In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.     

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae

 A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates. Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants. Received: 8 July 1996 / Accepted: 21 August 1996     

Light-harvesting proteins of diatoms: Their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids

Summary We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic -helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.     

A new member of the CAB gene family: structure,expression and chromosomal location of Cab-8, the tomato gene encoding the Type III chlorophyll a/b-binding polypeptide of photosystem I

We have previously reported the isolation and characterization of tomato nuclear genes encoding two types of chlorophyll a/b-binding (CAB) polypeptides localized in photosystem (PS) I and two types of CAB polypeptides localized in PSII. Sequence comparisons shows that all these genes are related to each other and thus belong to a single gene family. Here we report the isolation and characterization of an additional member of the tomato CAB gene family, the single tomato nuclear gene, designated Cab-8, which encodes a third type of CAB polypeptide localized in PSI. The protein encoded by Cab-8 is 65% and 60% divergent from the PSI Type I and Type II CAB polypeptides, respectively. The latter two are 65% divergent from each other. Only some short regions of the polypeptides are strongly conserved. The Cab-8 locus maps to chromosome 10, 9 map units from Cab-7, the gene encoding the Type II PSI CAB polypeptide. The Cab-8 gene contains two introns; the first intron matches in position the single intron in the Type II PSII CAB genes and the second intron matches in position the second intron in the Type II PSI CAB gene. Like other CAB genes, Cab-8 is light-regulated and is highly expressed in the leaf and to a lesser extent in other green organs.     

Sequence of the fourth and fifth Photosystem II Type I chlorophyll a/b-binding protein genes of Arabidopsis thaliana and evidence for the presence of a full complement of the extended CAB gene family

A second locus (Lhb1B) encoding Photosystem II Type I chlorophyll a/b-binding (CAB) polypeptides was identified in Arabidopsis thaliana. This locus carries two genes in an inverted orientation. The predicted sequences of the polypeptides encoded by these two genes show substantial divergence in their amino termini relative to each other and to the proteins encoded by the three Lhb1 CAB genes previously characterized [10], but little divergence within the predicted primary structure of the mature protein. DNA probes derived from seven additional types of tomato CAB genes, encoding chlorophyll a/b-binding polypeptides of several antenna systems of the photosynthetic apparatus, were tested against A. thaliana. Each of these hybridized in Southern blots to unique DNA fragment(s), demonstrating the existence of each of these different types of CAB genes in the genome of A. thaliana. The number of genes encoding each CAB type in A. thaliana was estimated to be similar to that of tomato.     

Nucleotide sequence of two cDNAs encoding fucoxanthin chlorophyll a/c proteins in the diatom Odontella sinensis

Two cDNA clones encoding fucoxanthin chlorophyll a/c-binding proteins (FCP) in the diatom Odontella sinensis have been cloned and sequenced. The derived amino acid sequences of both clones are identical, comparison of the corresponding nucleic acids reveals differences only in the third codon position, suggesting a recent gene duplication. The derived proteins are similar to the chlorophyll a/b-binding proteins of higher plants. The presequences for plastid import resemble signal sequences for cotranslational import rather than transit peptides of higher plants. They are very similar to the presequences of FCP proteins in the diatom Phaeodactylum, but different from the presequences of the -subunit of CF₀CF₁ of Odontella and the peridinin chlorophyll a binding proteins (PCP) of the dinoflagellate Symbiodinium.Abbreviations CAB chlorophyll a/b-binding protein - FCP fucoxanthin chlorophyll a/c-binding protein - fcp the respective FCP genes - LHC light-harvesting complex - PCP peridinin chlorophyll a-binding protein - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)⁺ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [³²P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [³²P] cDNA probe synthesized from poly(A)⁺ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.     

CHARACTERIZATION OF PHOTOSYSTEM I-ASSOCIATED POLYPEPTIDES FROM THE CHLOROPHYLL b-RICH ALGA TETRASELMIS SPP. (PLEURASTROPHYCEAE) AND OTHER CHLOROPHYTE ALGAE1

The phylogenetic distribution of photosystem I-associated polypeptides was assessed by immunoblotting algal thylakoid membrane polypeptides with antisera generated against the P700-chlorophyll a protein (CC I) and a photosystem I light-harvesting chlorophyll-protein (LHC Ib). Polypeptides cross-reacting with the CC I apoprotein were found in 20 species representing four classes of unicellular algae. Polypeptides sharing antigenicity with spinach LHC Ib were observed only in algal species containing chlorophyll b. Tetraselmis spp. (Pleurastrophyceae), rich in chlorophyll b (Chl a:b 1.2), exhibited marked heterogeneity in the composition of their CC I and LHC Ib cross-reactive polypeptides. When immunoblotted with antisera against CC I, all Tetraselmis clones examined exhibited a 25-kD polypeptide in greater abundance than the 58-kD CC I apoprotein characteristic of higher plants and other green algal thylakoids. Three Tetraselmis clones (RG 6, RG 11, and RG 12) exhibited an 81-kD polypeptide with strong antigenicity toward the LHC Ib antisera, in contrast to the 17- to 24-kD cross-reactive polypeptides found in spinach, green algae, and one Tetraselmis clone (RG 5). Associated with the unique photosystem I polypeptide composition in Tetraselmis spp., Chl: P700 ratios for the group are 2–5 times greater than those observed for higher plants or other green algae. The chlorophyll b enrichment, unusual composition of photosystem I cross-reactive polypeptides, and heterogeneity of these polypeptides within isolates of Tetraselmis might make this genus useful for investigations of the functional organization of chlorophyll b in light-harvesting systems. These features also support the view of an alternative phyletic origin for the Pleurastrophyceae.     

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.     

A Phylogenetic Assessment of the Eukaryotic Light-Harvesting Antenna Proteins, with Implications for Plastid Evolution

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment–protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin–chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin–chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes. Received: 25 February 1998 / Accepted: 13 May 1998     

Molecular characterization of two clusters of genes encoding the Type I CAB polypeptides of PSII in Nicotiana plumbaginifolia

A Nicotiana plumbaginifolia genomic library in the phage Charon 34 was used to isolate and characterize 7 full-length genes and part of an 8th gene encoding chlorophyll a/b-binding (CAB) polypeptides. These genes are arranged in two clusters. All the genes within the clusters are arranged in opposite orientation to their neighbours. The nucleotide sequences of two genes, one from each cluster, show that both genes, designated Cab-E and Cab-C, encode very similar proteins (95.9% of homology) corresponding to type I photosystem II polypeptides. Southern blot analysis suggests that at least 19 CAB genes encoding type I PSII CAB polypeptides are present in the N. plumbaginifolia genome. We also describe the presence within the N. plumbaginifolia genome of CAB genes encoding PSII type II CAB polypeptides and PSI type I CAB polypeptides. The sequences of the 5 flanking region of three different CAB genes (Cab-E, Cab-C, and CAB-F) were determined. Two of them (Cab-C and Cab-F) share extensive homology, whereas the Cab-E promoter shows homology to Cab-C and Cab-F only in a unique region extending from the CAAT box to the TATA box. This conserved sequence is also found in the same position in promoters of CAB genes encoding type I PSII polypeptides from other plant species.Abbreviations CAB chlorophyll a/b-binding protein - bp base pair(s) - kb 1 000 bp     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the L1637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.     

Alterations in chlorophyll a/b binding proteins in Solanaceae cybrids

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11–12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.     

Molecular characterization and genetic mapping of DNA sequences encoding the Type I chlorophyll a/b-binding polypeptide of photosystem I in Lycopersicon esculentum (tomato)

We report the isolation and characterization of a tomato nuclear gene encoding a chlorophyll a/b-binding (CAB) protein of photosystem I (PSI). The coding nucleotide sequence of the gene, designated Cab-6B, is different at eight positions from that of a previously isolated cDNA clone derived from the Cab-6A gene, but the two genes encode identical proteins. Sequence comparison with the cDNA clone revealed the presence of three short introns in Cab-6B. Genetic mapping experiments demonstrate that Cab-6A and Cab-6B are tightly linked and reside on chromosome 5, but the physical distance between the two genes is at least 7 kilobases. Cab-6A and Cab-6B have been designated Type I PSI CAB genes. They are the only two genes of this branch of the CAB gene family in the tomato genome, and they show substantial divergence to the genes encoding CAB polypeptides of photosystem II. The Type I PSI CAB genes, like the genes encoding PSII CAB proteins, are highly expressed in illuminated leaf tissue and to a lesser extent in other green organs.     

The novel light-harvesting pigment-protein complex ofMantoniella squamata (Chlorophyta): Phylogenetic implications

Summary A light-harvesting pigment-protein complex has been isolated fromMantoniella squamata (Micromonadophyceae, Chlorophyta) by nondenaturing polyacrylamide-gel electrophoresis. The complex runs as two bands of molecular weights 54,000 and 55,000. There are two constituent polypeptides of molecular weights 20,500 and 22,000. Antibodies were raised to the 20,500-dalton polypeptides from this complex and to the 24,500-dalton polypeptide from the analogous complex ofPedinomonas minor (Micromonadophyceae). The antibodies to theM. squamata polypeptide are specific for both polypeptides of theM. squamata light-harvesting complex, as well as for a 27,000-dalton polypeptide of undetermined function. The antibodies to theP. minor polypeptide are specific for polypeptide components of the light-harvesting complex of that alga. The antibodies specific for theM. squamata light-harvesting complex polypeptides do not cross react with any polypeptides ofP. minor thylakoid membranes, as demonstrated by crossed immunoelectrophoresis. Similarly, no polypeptides ofM. squamata thylakoids cross react with the antibodies specific forP. minor light-harvesting complex polypeptides. These results indicate that the light-harvesting complex ofM. squamata is structurally very different from that ofP. minor. In a survey of several land plants and green algae, including representatives of all classes of green algae, a light-harvesting complex homologous to that ofM. squamata was found only inMicromonas pusilla. All other organisms tested possessed a lightharvesting complex homologous to that ofP. minor. The evolutionary and taxonomic implications of the novelM. squamata light-harvesting complex are discussed.     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic -helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.This study is dedicated to Prof. Dr. W Rüdiger on the occasion of his 60th birthday     

The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

We have adapted the procedure for the isolation of PSII membranes from higher plants (D.A. Berthold et al., 1981, FEBS Lett. 134, 231–234) to the green algae Chlamydomonas reinhardtii. The chlorophyll (Chl)-binding proteins from this PSII preparation have been further separated into single Chl-binding polypeptides and characterized spectroscopically. Seven single polypeptides were shown to bind Chl a and Chl b. In particular, we demonstrate that polypeptides p9, p10 and p22, which had not been previously shown to bind Chl a and b, have characteristics similar to those of CP29, CP26 and CP24 from higher plants. We note, however, that p9 and p10 are phosphorylatable in C. reinhardtii, at variance with CP29 and CP26 from higher plants. Our data support the notion that the PSII antenna systems in C. reinhardtii and in higher plants are very similar. Therefore, studies on the organization and regulation of light-harvesting processes in C. reinhardtii may provide information of general relevance for both green algae and higher plants.Abbreviations Chl chlorophyll - IEF isoelectrofocusing - LHC light harvesting complex - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PS photosystem - RC reaction centre - SDS sodium dodecylsulfate We thank Dr. J. Olive (Institut Jacques Monod, Paris, France) for the electron-microscopy analysis, C. de Vitry (Institut de Biologie Physico-Chimique, Paris, France) for the kind gift of a PSII RC preparation and P. Dainese and M.L. Di Paolo (Universitá di Padova, Padova, Italy) for helpfull discussions. Professor Strasser and Elizbeth Scwartz (Université de Genova, Genova, Switzerland) are thanked for assistance in taking low-temperature fluorescence emission spectra. Roberto Bassi was recipient of a short-term fellowship from the European Molecular Biology Organization fellowship, during the early phases of the work.     

The tomato Cab-4 and Cab-5 genes encode a second type of CAB polypeptides localized in Photosystem II

The photosynthetic apparatus of plant chloroplasts contains two photosystems, termed Photosystem I (PSI) and Photosystem II (PSII). Both PSI and PSII contain several types of chlorophyll a/b-binding (CAB) polypeptides, at least some of which are structurally related. It has been previously shown that multiple genes encoding one type of PSII CAB polypeptides exist in the genome of many higher plants. In tomato, there are at least eight such genes, distributed in three independent loci. Genes encoding a second type of CAB polypeptides have been isolated from several plant species, but the precise location of the gene products has not been determined. Here we show that tomato has two unlinked genes encoding this second type and that this type of CAB polypeptide is also localized in PSII.     

The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

The role of transit peptides in intraorganellar targeting has been studied for a chlorophyll a/b binding (CAB) polypeptide of photosystem II (PSII) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) from Pisum sativum (pea). These studies have involved in vitro import of fusion proteins into isolated pea chloroplasts. Fusion of the CAB transit peptide to RBCS mediates import to the stroma, as evidenced by assembly of RBCS with chloroplast-synthesized large subunit (RBCL) to form holoenzyme. Similarly, fusion of the RBCS transit peptide to the mature CAB polypeptide mediates import and results in integration of the processed CAB protein into the thylakoid membrane. Correct integration was indicated by association with PSII and assembly with chlorophyll to form the light-harvesting chlorophyll a/b protein complex (LHCII). We interpret these results as evidence that the CAB transit peptide is functionally equivalent to a stromal-targeting sequence and that intraorganellar sorting of the CAB protein must be determined by sequences residing within the mature protein. Our results and those of others suggest that import and integration of CAB polypeptides into the thylakoid proceeds via the stroma.