Neil E. Hoffman

A Chromodomain Protein Encoded by the Arabidopsis CAO Gene Is a Plant-Specific Component of the Chloroplast Signal Recognition Particle Pathway That Is Involved in LHCP Targeting

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting–organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein–protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast–ribosomal association with chloroplast ribosomes.

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast‐encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast‐encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV‐activatable cross‐linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross‐linkers. With both cross‐linking approaches, the D1 ribosome nascent chain was photocross‐linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross‐link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.

Chloroplast FtsY, Chloroplast Signal Recognition Particle, and GTP Are Required to Reconstitute the Soluble Phase of Light-harvesting Chlorophyll Protein Transport into Thylakoid Membranes

The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43- and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, anEscherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport.

Chloroplast SecY is complexed to SecE and involved in the translocation of the 33-kDa but not the 23-kDa subunit of the oxygen-evolving complex.

SecY is a component of the protein-conducting channel for protein transport across the cytoplasmic membrane of prokaryotes. It is intimately associated with a second integral membrane protein, SecE, and together with SecA forms the minimal core of the preprotein translocase. A chloroplast homologue of SecY (cpSecY) has previously been identified and determined to be localized to the thylakoid membrane. In the present work, we demonstrate that a SecE homologue is localized to the thylakoid membrane, where it forms a complex with cpSecY. Digitonin solubilization of thylakoid membranes releases the SecY/E complex in a 180-kDa form, indicating that other components are present and/or the complex is a higher order oligomer of the cpSecY/E dimer. To test whether cpSecY forms the protein-conducting channel of the thylakoid membrane, translocation assays were conducted with the SecA-dependent substrate OE33 and the SecA-independent substrate OE23, in the presence and absence of antibodies raised against cpSecY. The antibodies inhibited translocation of OE33 but not OE23, indicating that cpSecY comprises the protein-conducting channel used in the SecA-dependent pathway, whereas a distinct protein conducting channel is used to translocate OE23.

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.

Distinct “Assisted” and “Spontaneous” Mechanisms for the Insertion of Polytopic Chlorophyll-binding Proteins into the Thylakoid Membrane

The biogenesis of several bacterial polytopic membrane proteins has been shown to require signal recognition particle (SRP) and protein transport machinery, and one such protein, the major light-harvesting chlorophyll-binding protein (LHCP) exhibits these requirements in chloroplasts. In this report we have used in vitro insertion assays to analyze four additional members of the chlorophyll-a/b-binding protein family. We show that two members, Lhca1 and Lhcb5, display an absolute requirement for stroma, nucleoside triphosphates, and protein transport apparatus, indicating an “assisted” pathway that probably resembles that of LHCP. Two other members, however, namely an early light-inducible protein 2 (Elip2) and photosystem II subunit S (PsbS), can insert efficiently in the complete absence of SRP, SecA activity, nucleoside triphosphates, or a functional Sec system. The data suggest a possibly spontaneous insertion mechanism that, to date, has been characterized only for simple single-span proteins. Of the membrane proteins whose insertion into thylakoids has been analyzed, five have now been shown to insert by a SRP/Sec-independent mechanism, suggesting that this is a mainstream form of targeting pathway. We also show that PsbS and Elip2 molecules are capable of following either “unassisted” or assisted pathways, and we discuss the implications for the mechanism and role of SRP in chloroplasts.

Biogenesis of a Photosystem I Light-Harvesting Complex (Evidence for a Membrane Intermediate)

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane inter-mediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/Bgl2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/Bgl2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the bio-genesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.

Reconstitution of Co-Translational Targeting of Polytopic Membrane Proteins to the Thylakoids in a Homologous Chloroplast Translation System

Chloroplasts in higher plants posses circular DNA containing 35 genes encoding for essential proteins located in the thylakoid membranes. The mechanisms of targeting and insertion of these proteins are unknown, despite their importance. Based on a number of observations in chloroplasts, it can be postulated that targeting and insertion of the polytopic chloroplast-encoded membrane proteins occurs co-translationally (1–3). Thus in order to reconstitute this targeting and insertion process, a homologous chloroplast in vitro initiation/translation system is required in which plasmid derived transcripts can be faithfully translated. The recent discovery of a translation system isolated from tobacco chloroplasts has opened up novel possibilities to address these important processes at a molecular level (4). In this paper, we have set out to evaluate the interaction of soluble stromal components cpSRP54 (5), cpSRP43 (6) and SecA (7) with the chloroplast encoded D1 protein, using this translation system. We show that ribosome D1 nascent chain complexes (D1 rncs) can be targeted very efficiently to the membrane and make functional interactions. In addition we show that cpRP54 interacts specifically with D1 rncs of defined length, implying a role for cpSRP54 in D1 biogenesis. To study the extent of conservation and mechanisms of the chloroplast targeting mechanisms with prokaryotes, we translated E.coli Leader peptidase (Lep) in the chloroplast system and attempted targeting Lep to the thylakoid membrane. Lep has two membrane spans and has served as a model protein to study targeting and insertion to the E. coli inner membrane (8–10) as well as to ER membranes (e.g.11). Lep is made without a cleavable signal sequence and its N- and C-terminal domains face the periplasmic side of the inner membrane (Fig. 4). In E.coli, insertion of Lep is SecA, SecY and SRP dependent (8–10).

Co-Translational Interaction and Targeting of the D1 Protein in a Homologous Chloroplast Translation System

The mechanisms of targeting and insertion of the chloroplast-encoded thylakoid proteins (about 35) are unknown, despite their importance. Based on a number of observations in chloroplasts, it can be postulated that targeting and insertion of the polytopic chloroplast-encoded membrane proteins occurs co-translationally (1–3). Thus in order to reconstitute this targeting and insertion process, a homologous chloroplast in vitro initiation/translation system is required in which plasmid derived transcripts can be faithfully translated. The recent discovery of a translation system isolated from tobacco chloroplasts has opened up novel possibilities to address these important processes at a molecular level (4). Using this system, we have observed that ribosome D1 nascent chains (rncs) interact with cpSRP54 (5), implicating this protein in D1 protein biogenesis. No interactions of rncs with cpSRP43 (6) or SecA (7) were detected, in absence of membranes. Furthermore, we found that D1 rncs were efficiently targeted to the thylakoid membrane, thereby demonstrating that the components required for targeting D1 to the membrane are contained in the tobacco translation extract.

Structure of chloroplast signal recognition particle and its role in chloroplast biogenesis

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that facilitates the co-translational insertion of proteins into the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. In prokaryotes, the SRP-RNA binds a single 54 kD polypeptide subunit, while in eukaryotes, five additional subunits are bound. A homologue of the 54 kD subunit of SRP was found in chloroplasts; it is 44% and 27% identical to SRP54 from E. coli and dog, respectively (1). This paper describes our progress in determining the structure of cpSRP and its role in the chloroplast. In particular we find that cpSRP, comprised of 43 and 54 kD subunits, plays a role in the biogenesis of light harvesting chlorophyll proteins (LHCP), the major proteins of the thylakoid membrane. LHCP form a large family of related proteins that have three to four transmembrane domains. They are synthesized in the cytoplasm, and are targeted to the thylakoid membrane through the stroma by a post-translational mechanism. Evidence is also presented that cpSRP54 functions independently of cpSRP43 in the co-translational targeting of chloroplast encoded proteins.