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Synthesis of Several Light-Harvesting Complex I Polypeptides Is Blocked by Cycloheximide in Symbiotic Chioroplasts in the Sea Slug, Elysia chlorotica (Gould): A Case for Horizontal Gene Transfer Between Alga and Animal?

JEFFREY J. HANTEN [1,2]

SIDNEY K. PIERCE [2][*]

Abstract. The chloroplast symbiosis between the ascoglossan (=Sacoglossa) sea slug Elysia chlorotica and plastids from the chromophytic alga Vaucheria litorea is the longest-lived relationship of its kind known, lasting up to 9 months. During this time, the plastids continue to photosynthesize in the absence of the algal nucleus at rates sufficient to meet the nutritional needs of the slugs. We have previously demonstrated that the synthesis of photosynthetic proteins occurs while the plastids reside within the diverticular cells of the slug. Here, we have identified several of these synthesized proteins as belonging to the nuclearencoded family of polypeptides known as light-harvesting complex I (LHCI). The synthesis of LHCI is blocked by the cytosolic ribosomal inhibitor cycloheximide and proceeds in the presence of chloramphenicol, a plastid ribosome inhibitor, indicating that the gene encoding LHCI resides in the nuclear DNA of the slug. These results suggest that a horizontal transfer of the LHCI gene fr om the alga to the slug has taken place.

Introduction

Most alga-animal symbioses are extracellular associations between two genetically distinct organisms. The alga is usually located extracellularly or enclosed within vacuoles inside the animal's cells. Rarer, but not uncommon, are intracellular symbioses occurring with intact algal chloroplasts that are captured by specialized cells within the animal. In particular, several species of ascoglossan (=Sacoglossa) (Opistobranchia) sea slugs capture intact, functional plastids from their algal food source and retain them within specialized cells lining the mollusc's digestive diverticula. This phenomenon has been termed chloroplast symbiosis (Taylor, 1970) or kleptoplasty (Clark et al., 1990). The sequestered plastids continue to photosynthesize for periods ranging from a few days to a few months, depending on the species (Greene, 1970; Hinde and Smith, 1974; Graves et al., 1979; Clark et al., 1990).

The longest such association, lasting as long as 9 months, is found in Elysia chlorotica (Gould), which obtains symbiotic plastids from the chromophytic alga Vaucheria litorea (C. Agardh) (West, 1979; Pierce et al., 1996). The association begins at metamorphosis of the slug from planktonic veliger to juvenile. In laboratory cultures, filaments of V. litorea must be present for metamorphosis to take place (West et al., 1984). Veligers home in, attach to the filaments, and metamorphose into juvenile slugs over the next 24 h. The juveniles eat the algal filaments and sequester the chloroplasts within one of at least two morphologically distinct types of epithelial cells lining the walls of the digestive diverticula (West et al, 1984). Once the plastids are sequestered, the slugs can sustain photosynthesis at rates sufficient to satisfy the nutritional needs for the complete life cycle of the slug, when provided with direct light and carbon dioxide (Mujer et al., 1996; Pierce et al., 1996).

Even in nature the slugs obtain most of their energy from photosynthesis (West, 1979).

The longevity of this relationship in E. chlorotica makes it especially interesting. Photosynthesis requires the continuous synthesis of a variety of chloroplast proteins because many of them, including those used in light harvesting, are rapidly degraded and must be replaced (Greenberg et al., 1989; Mattoo et at., 1989; Barber and Andersson, 1992; Wollman et at., 1999). Furthermore, photosynthesis requires the interaction of as many as 1000 proteins, only about 13% of which are coded in the plastid genome (Martin and Herrmann, 1998). In the plant cell, substantial nuclear input is required to sustain photosynthetic function, in the form of direct coding of the proteins as well as providing the means for their intracellular transport and regulation (Berry-Lowe and Schmidt, 1991; Wollman et at., 1999). Considering the level of nuclear and extra-plastid input required, it is not surprising that the longevity of the plastids in most kleptoplastic slugs is relatively short. However, several photosynthetic protei ns are synthesized in the sequestered plastids of E. chlorotica (Pierce et at., 1996), including the large subunit of RuBisCO, Dl, D2, CP43, cyt land others (Pierce et at., 1996; Mujer et al., 1996; Green et al., 2000). Although all of the synthesized plastid proteins identified to date are plastid encoded (Mujer et at., 1996; Pierce et at., 1996; Green et at., 2000), two groups of synthesized plastid proteins can be distinguished pharmacologically: those inhibited by cycloheximide (CHX), an 80S cytosolic ribosome inhibitor (Obrig et at., 1971), and those inhibited by chloramphenicol (CAP), which inhibits protein synthesis on 70S plastid and mitochondrial ribosomes (Lamb et at., 1968; Stone and Wilke, 1975).

Because the inhibition by CHX suggests that the genes for several plastid proteins must reside in the nuclear DNA, we have done some experiments to identify these proteins and test that possibility. Our present study reports the identification of several of the CHX-blocked proteins as members of the light-harvesting complex I (LHCI), a family of pigment-binding proteins responsible for collecting radiation energy from sunlight and transferring it to photosystem I (PSI). LHCI proteins are encoded by the Lhca genes in the nuclear genome of all the plants and algae whose genomes have been examined (Jansson, 1994, 1999; Green and Durnford, 1996; Durnford et at., 1999; Wollman et at., 1999). This result suggests that the LHCI genes have been somehow transferred from the algal nucleus to the slug's DNA.

Materials and Methods

Animals and alga

Specimens of Elysia chlorotica were collected in both the spring and fall from an intertidal marsh near Menemsha Pond on the island of Martha's Vineyard, Massachusetts. The slugs were maintained in 10-gallon aquaria at 10 [degrees]C in aerated, artificial seawater (ASW; Instant Ocean, 925-1000 mosm) on a 16/8-h light/dark cycle (GE cool-white fluorescent tubes, 15 W).

Sterile cultures of Vaucheria litorea were maintained in enriched ASW (400 mosm) [modified from the F/2 medium (Bidwell and Spotte, 1985)]. The alga was grown at 20 [degrees]C on a 16/8-h light/dark cycle (GE cool-white fluorescent tubes; 40 W), and the medium was changed weekly.

Inhibitor treatments and plastid protein labeling

All reagents used were molecular bio-grade (DNase-, RNase-, and protease-free) purchased from Sigma unless otherwise noted. Effective concentrations of CHX and CAP were determined empirically with initial dose-response curves (Pierce et at., 1996). CHX (2 mg [ml.sup.-1] was used to inhibit protein synthesis on 80S cytosolic ribosomes; CAP (160 [micro]g [ml.sup.-1]; stock concentration 50 mg [ml.sup.-1] in absolute ethanol) was used to inhibit translation on 70S plastid and mitochondrial ribosomes. Two to four slugs, total wet weight about 1.25 g, were placed into glass scintillation vials containing ASW (1000 mosm) and the appropriate inhibitor, and incubated under intense light (150 W, GE Cool Beam incandescent indoor flood lamp) at 20 [degrees]C in a gently agitating water bath. After 1 h, 20 [micro]Ci [ml.sup.-1] [[S.sup.35]]-methionine (0.7 MBq [ml.sup.-1], trans-[[S.sup.35]]-methionine, ICN) was added, and the slugs were incubated for an additional 6 h, previously demonstrated to provide ampl e time to incorporate radioactive label into the plastid proteins (Pierce et at., 1996). Additional slugs were incubated in 0.025% ethanol/ASW (v/v) solution plus [[S.sup.35]]-methionine to serve as a control for the carrier in CAP treatments.

Chloroplast isolation and protein separation

Chloroplasts were isolated from slugs by using a centrifugation protocol. The slugs were homogenized in the presence of the mucolytic agent N-acetyl-cysteine (500 mM), and the homogenate was filtered successively through cheesecloth, Miracloth (Calbiochem), and then nylon mesh (60 [micro]m to 10 [micro]m) to remove large debris and the copious amount of mucus the animals produce. The plastids were purified on a pre-formed, 25% Percoll (v/v) gradient, which provides a very pure fraction containing large numbers of intact plastids (Pierce et at., 1996). In this experiment, the lowest green band containing labeled plastids was isolated from the gradient by using a flamed Pasteur pipette, and residual Percoll was removed by centrifugation. The purified chloroplast pellets were resuspended, lysed by freezethawing, and stored at -20[degrees]C until use. The incorporation of radioactive label was determined by a liquid scintillation counter (Beckman LS60001C), and the protein content was determined using the modif ied Lowry assay (Peterson, 1977). The resulting specific activity was calculated as counts per minute (cpm) [([micro]g protein).sup.1]. Chlorophyll content was determined by extracting the pigment in 80% acetone, then measuring the extract absorbance spectrophotometrically at 652 nm. The results were calculated as micrograms per microliter according to standard equations (Joyard et al., 1987).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography was used to assess the effects of CHX and CAP on the pattern of protein synthesis. The plastid lysates obtained from the above procedure were boiled for 2 min in Tris-HCl (pH 6.8)-10% SDS (w/v) buffer containing 5% [beta]-mercaptoethanol [beta]-ME) (v/v). The solubilized proteins were loaded in equal amounts onto 15% SDS-polyacrylamide gels and separated by electrophoresis (Laemlli, 1970). The gels were stained with Coomassie brilliant blue, dried, and exposed to film (Kodak Biomax MR) for 2 to 30 days at -80[degrees]C, depending on the level of radioactive label present. Approximate molecular masses of the proteins were determined by comparison to the migration distances of known molecular weight standards (BioRad, broad-range kaleidoscope) run in adjacent lanes on each gel.

Immunoblot identification of plastid proteins

After the plastid isolation and protein separation via SDS-PAGE as described above, the proteins were electrophoretically transferred (30 V, 4 [degrees]C, overnight) to PVDF membranes (Immobilon-P; Millipore) (Towbin et al., 1979). As additional controls, V. litorea chloroplasts [isolated and purified using a 30% to 75% Percoll step gradient as previously described (Pierce et al., 1996)] and thylakoids from the red alga Porphyridium cruentum (generously donated by Professor Elisabeth Gantt, University of Maryland), were lysed, and the proteins were separated electrophoretically and transferred to membranes as above. The membranes were blocked with 5% (w/v) dehydrated milk dissolved in Tris-buffered saline (TBS) (Tris-base 50 mM, NaCl 0.9%, pH 7.5) for 1 h at room temperature, washed twice in TBS for 10 mm, and treated with primary antibody for 1 h. In this case, the primary antibody was a polyclonal antibody to LHCI which was produced in a rabbit using a 22-kDa, recombinant LHCI polypeptide produced from a c lone of the LhcaRI gene of P. cruentum (Grabowski et al., 2000) (also provided by Professor Gantt) ["RI" indicating it is a rhodophyte gene (Tan et al., 1997a)] as the antigen combined with Freund's adjuvant in a standard immunization procedure. After binding of the primary antibody, the membranes were washed twice as above and incubated with secondary antibody, anti-rabbit conjugated hydrogen peroxidase, for 1 h. After washing, the bands were visualized with a 4-chloro-1-napthol and hydrogen peroxide reaction according to manufacturer's instructions. The immunolabeled western blots were exposed to film as described above to identify the coincidence of antibody binding and radioactive incorporation in the presence of each inhibitor.

As a control to confirm that the CAP was blocking plastid-directed protein synthesis and that CHX was not, parallel measurements were run to monitor cytochrome f (cytf) synthesis. Earlier experiments conducted on E. chlorotica have demonstrated that cyt f is synthesized in the slugs and is encoded in the plastid DNA (Green et al., 2000). Thus, if CHX and CAP are working as expected, their effect on cyt f and any nuclear-encoded proteins should be opposite. Anti-cyt f, raised to P. cruentum cyt f, was also a gift of Professor Gantt.

Immunoprecipitations

Immunoprecipitations were conducted to confirm the identity of the radioactive immunolabeled bands on the western blots, using a modified version of the protocol previously used to precipitate proteins from isolated E. chlorotica plastids (Pierce et al., 1996). Plastid proteins were solubilized in lysing buffer (10 mM Tris-HC1, 10 mM EDTA, 150 mM NaCl, 1 mM PMSF, 1% (v/v) Nonidet P-40, pH 8.0), using equal amounts of chlorophyll per sample, mixed with a small amount of Protein-A Sepharose beads to eliminate nonspecific binding, and incubated on ice with occasional agitation. The beads were removed by centrifugation and discarded, the supernatant was saved, and the appropriate antibody was added to the lysate and rotated overnight (4 [degrees]C). Protein-A beads, swelled in washing buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCI, 1 mM PMSF, 0.1% (v/v) Nonidet P-40, pH 8.0), were added the following morning and rotated (3 h, room temperature). The antigen-antibody-protein-A Sepharose bead complexes were washed several times in washing buffer and removed by centrifugation. In the case of cyt f, the antigen-antibody-protein-A Sepharose bead complexes were resuspended in 10.0 M urea, 10% SDS (w/v), 5% [beta]-ME (v/v), pH 12.5, and boiled for 10 mm to liberate the cyt f antigen. The solution was centrifuged, the supernatant was removed, and the beads were discarded. The supernatant proteins were separated by SDS-PAGE as described above, and the gel was autoradiographed.

The LHCI antibody-antigen complex could not be broken efficiently with any treatment, which prevented the visualization of the labeled LHCI proteins via SDS-PAGE. Although this was unexpected, it is not unusual and may have been caused by a number of factors. The presence of several different LHCI polypeptides with varying isoelectric points, ranging between 4.5 and 9.5 (De Martino et al., 2000), makes it very difficult to create optimal reaction conditions for each one. The polyclonal antibody molecules bind to all the LHCI polypeptides as well as to each other, creating a large antigen-antibody complex with a core inaccessible to the chemicals necessary to liberate the antigen. Very few researchers have attempted LHC immunoprecipitations because of the pitfalls involved in precipitating inner-membrane proteins (Anderson and Blobel, 1983). Instead, other protocols have been designed using mild detergents to extract intact photosystem holocomplexes from the thylakoids, followed by protein separation on sucro se density gradients (Fawley and Grossman, 1986; Buchel and Wilhelm, 1993; Wolfe et at., 1994; Schmid et at., 1997). These isolations require large amounts of starting material (Schmid et at., 1997) that greatly exceed what is available to us in the slugs. So, instead, we used the LHCI antibody to demonstrate that LHCI had incorporated radioactivity.

Following the procedure described above, the protein A Sepharose beads were reacted with anti-LHCI and then with a radiolabeled plastid protein extract. The antigen-antibody-protein-A Sepharose bead complexes were repeatedly washed by centrifugation until the radioactivity in the supernatant was reduced to background. The washed antigen-antibody-protein-A Sepharose bead complexes were resuspended in optifluor (Packard), and radioactivity was determined by a scintillation counter. Controls for nonspecific binding to protein-A Sepharose beads were conducted with the same procedure, but without the addition of the LHCI antibody. Counts per minute resulting from nonspecific binding were subtracted from experimental values for each inhibitor treatment and controls, and the final data were converted to cpm [([micro]g chlorophyll).sup.-1] [([micro]g protein).sup.-1]. The normalized data were averaged and expressed in terms of percent of control for each inhibitor.

Results

Plastid protein synthesis and identification

The Coomassie-stained SDS-PAGE gels of protein extracts from isolated slug plastids were similar to controls regardless of the inhibitor present, either CHX or CAP, indicating no difference in the protein composition of the plastids after treatment (Fig. 1). However, autoradiograms of SDS-PAGE gels of plastid proteins extracted from slugs incubated in the presence of [[S.sup.35]]-methionine indicate that very different patterns of protein synthesis occur in the slugs between controls and inhibitors as well as between inhibitors (Fig. 2). CHX has a profound effect on protein synthesis, preventing synthesis of the majority of the protein bands labeled in the absence of inhibitor (Fig. 2, CON), whereas the synthesis of many more labeled bands occurs in the presence of CAP. Furthermore, these protein bands differ from those visualized in the CHX treatments (Fig. 2).

Verification of inhibitor effects

Cyt f antibodies reacted with a protein band synthesized in the presence of CHX on western blots at approximately 36 kDa (Fig. 3). Immunoprecipitations using anti-cyt f identify a band with a molecular weight corresponding to cytf, confirming its identity (Fig. 4). Autoradiograms of the same gels show [[S.sup.35]]-methionine incorporation into cyt f in the presence of CHX, but not in the presence of CAP (Fig. 5).

The anti-LHCI we made to Porphyridium cruentum recombinant LHCI recognized both the recombinant LHCI antigen (Fig. 5A, lane l) and the LHCI polypeptides from P. cruentum thylakoids (Fig. SB, lane 2). Six polypeptide bands were identified in P. cruentum, ranging in approximate molecular weights from 19 to 24 kDa (Fig. 5B, lane 2), sizes consistent with those previously described for the LHCI polypeptides in this species (Tan et at., 1995). The antibody bound onto western blots of plastid proteins from Vaucheria titorea and Etysia chlorotica, with or without the CHX and CAP treatments (Fig. 5C, lanes V. lit., CON, CHX, CAP). As expected, the six polypeptide bands bound by the anti-LHCI in V. litorea and E. chlorotica plastids have a slightly greater size range--18 to 32 kDa--than those identified in P. cruentum. These same antibody-labeled bands from E. chlorotica plastid proteins incorporate radioactive label in the presence of CAP, but incorporation is blocked by the presence of CHX (Fig. 6).

The amount of radiolabel precipitated by anti-LHCI from the slug plastid extracts following CHX treatment is only 2% of the control level, indicating a reduction in LHCI synthesis (Fig. 7). In contrast, the LHCI proteins in CAP-treated slugs incorporated [[blank.sup.35]-methionine at 92% of control rates, more than 40-fold higher than the level found in CHX treated animals (Fig. 7).

Discussion

LHCI, a family of plastid polypeptides essential for photosynthesis, is synthesized while Vaucheria litorea chloroplasts reside within the cells of the digestive diverticula of Elysia chlorotica. In addition, our data indicate the LHCI polypeptides are probably the products of genes located in the host-cell nuclear genome because their synthesis is inhibited by the cytosolic ribosome inhibitor, CHX, but not by the presence of the plastid ribosome inhibitor, CAP. This remarkable result would not be surprising in a plant or algal species since the LHCI polypeptide family's genes, Lhcal-Lhca6, reside in the nuclear DNA of all plants and algae examined to date (Jansson, 1994; Green and Dumford, 1996; Durnford et al., 1999; Jansson, 1999; Wollman et at., 1999). However, the synthesis of LHCI directed by an animal's genome indicates that genes have been transferred into the slug DNA.

Although surprising, the site of synthesis and the identification of LHCI seem to be without question as long as inhibitor and antibody specificity are not problems. Both CHX and CAP have been used in a wide array of studies, and their sites of action are well established. In fact, they have been used, exactly as we have done here, to establish that the site of synthesis of the "light harvesting chlorophyll protein" (=LHCI) occurs on 80s cytoplasmic ribosomes in Phaeodactylum tricornuturm (Fawley and Grossman, 1986).

There are several reasons to conclude that our antibody is specific. We raised the antibody against the red alga LHCI not only because it was available, but also because the chromophytes, the taxonomic group of V. litorea, probably arose through a secondary symbiosis from a red alga (Rieth, 1995; Green and Durnford, 1996; Palmer and Deiwiche, 1996; Martin and Herrmann, 1998; Delwiche, 1999). Furthermore, Porphyridium cruentum LHCI possesses both sequence homologies and immunological relatedness to the chromphytic light-harvesting proteins (Wolfe et at., 1994; Rieth, 1995; Tan et al., 1997b). Thus, a polyclonal antibody raised to a rhodophyte LHCI should have a good chance of specifically recognizing the LHCI polypeptides in V. litorea. Our results indicate that the anti-LHCI binds the P. cruentum recombinant LHCI, the antigenic source for the antibody, as well as all six of the native P. cruentum LHCI proteins (Tan et at., 1995; Grabowski et at., 2000) in control immunoblots of extracted thylakoids. The anti -LHCI immunoblots of E. chiorotica and V. litorea also identified six protein bands with a greater size range than the LHCI proteins identified in P. cruentum. Those bands are consistent with the sizes of LHCI polypeptides from many species (Gantt, 1996; Jansson, 1999; Wollman et at., 1999), and no other bands were labeled by the antibody. Seeing six LHCI proteins is not surprising, because LHCI is typically found in multiple homologs in algae, ranging from two in one species of Xanthophyceae (Buchel and Wilhelm, 1993) to at least six paralogs in some rhodophytes (Tan et al., 1995), and as many as eight in the chromophyte Heterosigma carterae (Dumford and Green, 1994). With few exceptions [such as in Euglena gracilis (Jansson, 1994)], each is encoded by a separate, nuclear gene belonging to the Lhc super-gene family (Jansson, 1999). Thus, location of the gene aside, the presence of six LHCI proteins in the endosymbiotic plastids in E. chiorotica is not surprising.

It seems clear that each of the bands immunodecorated by anti-LHCI corresponds to a single LHCI polypeptide and not a dimer. LHCI dimers can result from their association with other LHC proteins and their respective photosystems in situ, and they do not always readily dissociate under the denaturing conditions of SDS-PAGE (Tan et al., 1995). If LHCI dimers were present here, they should have minimum molecular weights of about 36 kDa, corresponding to double the molecular weight of the smallest immunolabeled band. However, the largest of the six immunolabeled bands present in the gels is about 32 kDa, seemingly too small to be an LHCI dimer.

Other dimers might form with a number of photosystem I (PSI) proteins due to the close association of LHCI with the PSI subunits that compose the PSI-LHCI holocomplex (Wollman et al., 1999; Jansson, 1999). This also does not seem to be the case here. Anti-PSI, raised against the cyanobacteria PSI holocomplex (again, courtesy of Professor Gantt), binds a single 10-kDa protein band on western blots of E. chlorotica plastid proteins (data not shown). The combination of this PSI polypeptide with any of the three smaller bands (18-20 kDa) that react with the anti-LHCI could form a dimer with molecular weights comparable to each of the three larger polypeptides (28-32 kDa). However, since anti-PSI and anti-LHCI do not co-label any bands, an LHCI-PSI dimer is unlikely.

An additional possibility might be that one of the bands could be another LHC-type protein possessing immunological similarities to LHCI, such as the fucoxanthin chlorophyll a/C binding proteins (FCPC) found in chromophytes or light-harvesting complex II (LHCII) proteins. In fact, our previous work has demonstrated the presence of FCPC in plastids of both E. chiorotica and V. litorea. However, the size of the FCPC protein identified there does not correspond to the weights of the proteins bound by the anti-LHCI used here (Pierce et al., 1996; Green et al., 2000). Furthermore, previous attempts to demonstrate FCPC synthesis with radioactive labels in the slugs have not yielded positive results (Pierce et al., 1996).

The LHCII family of polypeptides is closely related to LHCI, performing similar functions in photosystem II to those performed by LHCI in PSI. The LHC II genes are in the same nuclear-encoded Lhc super-gene family (Jansson, 1999) and share sequence homologies with those genes encoding LHCI (Durnford et al., 1999; Jansson, 1999; Wollman et al., 1999). There is, however, a clear separation in the phylogenies of LHCI and LHCII (Dumford et al., 1999), indicating some degree of dissimilarity between the two proteins. Nevertheless, the possibility seems to remain that the proteins bound by our antibody could be from LHCII.

Of the LHCII components, CP24, CP26, and CP29 contain the most sequence similarities to the LHCIs (Green and Durnford, 1996) and have molecular weights, 25-30 kDa (Wollman et al., 1999), that roughly correspond to those of the three largest polypeptides identified in our anti-LHCI immunoblots of E. chlorotica and V. litorea plastid proteins (28-32 kDa), which appear to be slightly larger than most LHC proteins in chromophytes (Green and Durnford, 1996). An LHCII antibody derived from pea (generously donated by Dr. Kenneth Cline, University of Florida) was unreactive in our immunoblotting protocol (data not shown). This result seems to indicate that the polypeptides are not LHCII, but since the similarity between the green plant and chromophyte LHC proteins is relatively low (Green and Durnford, 1996; Durnford et al., 1999), we probably cannot completely eliminate the possibility that the anti-LHCI is binding LHCII polypeptides. However, just like LHCI, all of the LHCII genes are nuclear encoded in the plants and algae where they have been found (Jansson, 1994, 1999; Wollman et al., 1999), and even if we have identified LHCII, the conclusion is still the same: that an algal LHC gene has been transferred to the DNA of the slug.

The immunoprecipitations provide additional evidence that the LHCI polypeptides are being synthesized on the cytoplasmic ribosomes in the slug. The high amount of radioactivity precipitated by the antibody in the presence of CAP compared to that precipitated in the presence of CHX demonstrates that the proteins recognized by the anti-LHCI are indeed synthesized in the slugs. Since the amount of radioactivity incorporated varied from slug to slug and from experiment to experiment, we had to normalize the immunoprecipitation data as percent of control in order to compare them. However, in a typical experiment, the values for the amount of radioactive material incorporated into the precipitate in the presence of CAP ranged from 5000 to 25,000 cpm, whereas those in the presence of CHX ran from 150 to 400 cpm, which may give a clearer picture of the level of material bound by the antibody.

The results of the pharmacological experiments, the immunoblots, and the immunoprecipitations, taken together, provide substantial evidence that LHCI is the identity of some of the plastid proteins that are synthesized in the presence of CAP. The inhibition of LHCI synthesis by CHX suggests that the algal Lhca genes have somehow been transferred to the slug.

To be certain that a gene transfer has occurred, direct evidence of the gene in the genomic DNA of the slug must be found, and we are pursuing this confirmation. However, in addition to the results presented here, other circumstantial evidence for the transfer of the LHCI genes between alga and slug is available in several characteristics of the association. First, although the turnover rate of LHCI in E. chiorotica is unknown, the fact that it is synthesized indicates that it is not an unusually robust protein--LHCI replacement is necessary for plastid function to proceed. Second, Lhca genes have not been found in the plastid genomes of any organism (Durnford et al., 1999), including other Vaucheria species (Linne von Berg and Kowallik, 1992). Of course, if LHCI were present in the plastid genome, it would be synthesized with CHX present, as is the case with the cyt controls; but it is not. Third, the V. litorea plastid genome is 119.1 kb (Green et al., 2000), which is similar in size to those of other alga e, including V. sessilis and V. bursata (Linne von Berg and Kowallik, 1988, 1992), but small relative to those of other plants (Martin and Herrmann, 1998). Even though the plastid genomes of chromophytic algae have a greater coding capacity, relative to their size, than other algae because of fewer introns and inverted repeats (Rieth, 1995), they are too small to carry sufficient genetic information to encode all of the enzymes required for photosynthesis and plastid protein targeting. Fourth, transfer of algal DNA remnants or a nucleomorphtype structure during plastid capture seems unlikely. To date, nucleomorphs have been found only in the Cryptophyta and Chlorarachniophyta (Delwiche, 1999; Zauner et al., 2000) and have not been identified in any chromophyte (Maier et al., 1991; Delwiche, 1999). Although DNA of this type would probably be transcribed on nucleomorph 80S ribosomes (Douglas et al., 1991) and blocked by CHX, neither substantial electron microscopy (Kawaguti and Yamasu, 1965; Graves et al., 1979 ; Mujer et al., 1996) nor molecular testing (Green et al., 2000) has so far produced evidence for either nucleomorphs or algal nuclear remnants in E. chlorotica. Furthermore, if algal DNA remnants were present somewhere in the slug cells, the likelihood is remote of their containing the correct genes and being present in all of the plastid-containing cells in all of the slugs in the populations year after year. Finally, others have suggested that some of the proteins necessary to maintain photosynthesis may be encoded in the mitochondrial genome and are redirected to the chloroplast (Rumpho et al., 2000). Although dual targeting of proteins has been demonstrated in Arabidopsis (Chow et al., 1997; Menand et al., 1998), it seems highly unlikely with LHCI. LHCI has never been found associated with mitochondria in any organism; and CAP, which inhibits the mitochondrial ribosomes in addition to those associated with the plastids, would prevent its synthesis anyway.

The horizontal transfer of DNA from the endosymbiont to the nucleus of the host cell provides the basis for the theory of the endosymbiotic origin of eukaryotic organelles. This movement of the symbiont's genes to the host enabled the host to incorporate the organelle' s function into its own biochemistry and to faithfully replicate it in subsequent generations. The remnants of eubacterial genes in the mitochondrial and plastid genomes of modern eukaryotes probably resulted from such events (Martin and Herrmann, 1998). Most of the discussions regarding the evolution of plastids focus on the horizontal gene transfer resulting from the primary endosymbiotic event in which a primitive prokaryote engulfed a cyanobacteria (Palmer, 1993; Reith, 1995; Palmer and Delwiche, 1996; Martin et al., 1998; Tengs et al., 2000). Other hypotheses propose a secondary endosymbiosis, probably involving a eukaryote that engulfed a red or green alga (Gibbs, 1981; Palmer and Delwiche, 1996; Martin et al., 1998; Zhang et al., 1999; Delwiche, 1999; Tengs et al, 2000), that produced the plastids of the chromophytic algae and their relatives. In many of these cases, the identity of the initial host, symbiont, or both is unknown. In the case of E. chlorotica and V. litorea, the origin of LHCI is known; if the gene has been transferred, the transfer occurred between two multicellular eukaryotes and represents a case of tertiary endosymbiosis.

Finally, the mechanism by which such a gene transfer could occur may be found in the viruses that appear in each generation of the slugs at the end of their life cycle. The viruses have several features in common with Retroviridae and seem to be endogenous (Pierce et al., 1999). Retroviruses are capable of transferring genes between organisms; if they are incorporated in the germ cells, they are transferred to the subsequent generations as Mendelian genes (Scharfman et al., 1991). Thus, resolving the relationships between the slugs, alga, plastids, and viruses may have profound implications for both cell and evolutionary biology.

Acknowledgments

Research support was provided by a National Science Foundation award (IBN-9604679) to SKP. We thank Elisabeth Gantt and Beatrice Grabowski for their helpful suggestions.

(*.) To whom correspondence should be addressed. E-mail: pierce@chumal.cas.usf.edu

Abbreviations: CAP, chloramphenicol; CHX, cycloheximide; FCPC, fucoxanthin chlorophyll a/c binding proteins; LHC, light-harvesting complex; PSI; photosystem I.

(1.) Department of Biology, University of Maryland, College Park, Maryland 20742; and

(2.) Department of Biology, University of South Florida, Tampa, Florida 33620

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Author:HANTEN, JEFFREY J.; PIERCE, SIDNEY K.
Publication:The Biological Bulletin
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Date:Aug 1, 2001
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