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American Society of Plant Biologists Loss of Albino3 Leads to the Specific Depletion of the Light-Harvesting System
a Departments of Molecular Biology and Plant Biology, University of Geneva, Quai Ernest Ansermet 1211, Geneva 4, Switzerland 2 To whom correspondence should be addressed. E-mail jean-david.rochaix{at}molbio.unige.ch; fax 41-22-7026868
The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b6f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.
The biogenesis of the photosynthetic complexes of the thylakoid membrane depends on the concerted interactions of the chloroplast and nuclear genetic systems. Genetic and biochemical studies in Chlamydomonas, maize, and Arabidopsis have revealed a large number of nucleus-encoded factors that are involved in transcriptional and post-transcriptional steps of chloroplast gene expression (Barkan and Goldschmidt-Clermont, 2000  ). Once proteins of the photosynthetic apparatus have been synthesized, they need to be targeted to the thylakoid membrane, inserted into or translocated through the membrane, and assembled into functional complexes.
At least four pathways for thylakoid membrane insertion and translocation have been characterized that display unique energy and stromal protein requirements (Keegstra and Cline, 1999
The light-harvesting complex protein (LHCP) integration pathway shares several features with the GTP-dependent signal recognition particle (SRP) system of the endoplasmic reticulum and bacteria (Li et al., 1995
Together with GTP and the chloroplast homolog FtsY, an Escherichia coli protein necessary for E. coli SRP function, the transit complex promotes efficient integration of LHCP into the thylakoid membrane (Tu et al., 1999
Several factors specific for the assembly of photosynthetic complexes have been identified. They include BtpA (Bartsevich and Pakrasi, 1997
Studies with yeast mitochondrial protein export have shown that the inner membrane protein Oxa1p is an essential component for integrating a subset of inner membrane proteins encoded by nuclear and mitochondrial DNA (Bonnefoy et al., 1994
Recently, it was shown that treatment of thylakoid membranes with an Alb3 antibody interferes with the integration of Lhcb1, the major light-harvesting chlorophyll binding protein (Moore et al., 2000 To test the role of Alb3 in vivo, we have characterized a mutant of Chlamydomonas, ac29-3, in which the Alb3.1 gene is inactivated. This mutant is principally deficient in LHC. The level of PSII also is reduced, especially in dark-grown cells. The Alb3.1 protein appears to form two distinct complexes, a lower molecular mass complex of a size similar to that of LHC and a high molecular mass complex of unknown function. Chlamydomonas contains a homolog of Alb3.1 that appears to play only a modest role in the integration of LHC in the thylakoid membrane, based on the phenotype of the ac29 mutant.
Characterization of ac29 Mutants The first two mutants affected at the AC29 locus of Chlamydomonas, CC-44 and CC-45, were identified as yellow, leaky, acetate-requiring mutants (Levine and Goodenough, 1970 ). This locus is linked tightly to that of the MATING-TYPE (MT), which was cloned and characterized previously (Ferris and Goodenough, 1994; Ferris, 1995). Several new mutants affected at the AC29 locus were isolated during a search for large deletions in the MT locus (P. Ferris, unpublished results). A diploid having a prototrophic green mt- phenotype was constructed from CC-1336 (nic7 ac29-2 mt- pf14) and CC-1067 (++ mt+ +). Gamma irradiation of the diploid generated yellow and/or nicotinamide-requiring mutant strains carrying new mutations at these loci. Six yellow nicotinamide prototrophs (acy9, acy10, acy20, acy25, acy27, and acy32) were obtained that have DNA rearrangements in the region marked by the ac29-2 allele in Figure 1. DNA gel blot analysis of four of these mutants and their diploid parental strain is shown in Figure 2A.
The DNAs were digested with BglII and probed with the acB probe (Figure 1). The probe hybridizes to two fragments of the parent, one (1.3 kb) from the ac29-2 mt- chromosome and the other (2.2 kb) from the AC29 mt+ chromosome. The acy9 strain shows no change within the BglII fragments but has an insertion larger than 10 kb farther downstream (Figure 1 and data not shown). The acy27 and acy10 strains carry a small insertion and a deletion, respectively, within the 2.2-kb BglII fragment. These changes in the two mutants localize between the BstEII and SfiI sites (Figure 1 and data not shown). The only change detectable in the acy25 strain is the loss of the SfiI site. Figure 2B shows DNA gel blots obtained after digestion with SacI from the parent and the acy9, acy20, and acy32 mutants. The mt- and mt+ chromosomes yield fragments of 7.2 and 10.8 kb, respectively. In the mutants, the mt+ fragment has been replaced by smaller fragments. Additional DNA gel blots revealed that acy9 and acy20 contain large insertions (>10 kb) with several internal SacI sites, resulting in SacI fragments smaller than in the original, whereas acy32 carries a 3-kb deletion (data not shown). The approximate locations of the insertions and the deletion are indicated in Figure 1.
Identification and Structure of the AC29 (Alb3) Gene
Chlamydomonas ESTs indicate that Alb3.1 mRNAs are polyadenylated at multiple locations. The position of Alb3.1 is consistent with previous transformation data (Ferris, 1995 ). Thus, the green phenotype of the ac29-2 mutant was restored by transformation with the pAc5.8 plasmid, which terminates within the 3' untranslated region, but not by transformation with the HJ7 phage, which lacks the final exon (Figure 1). The location of Alb3.1 also is consistent with the observation that phages HF3 and CF2a were able to rescue the ac29-2 mutant by transformation (Ferris, 1995). Further evidence that the AC29 locus corresponds to the Alb3.1 gene is provided by the ac29-2 mutation in the third exon of Alb3.1. Restriction fragment length polymorphism analysis had revealed polymorphic BglII-XbaI tandem sites in the ac29-2 allele of CC-44. A PCR product spanning the mutant site obtained with primers 1 and 5 (Figure 1) was cloned and sequenced. The mutation consists of a single C-to-T base change that produces a stop codon and creates two novel restriction sites, BglII and XbaI (Figure 4).
It is possible that some other changes in the ac29-2 allele also are responsible for the phenotype, because the entire allele was not sequenced. However, examination of three green revertants by DNA gel blot analysis revealed that they had retained the BglII site but had lost the XbaI site. The only change in the sequence of the revertants relative to ac29-2 was within the XbaI site that restored the Alb3.1 open reading frame (Figure 4). Analysis of the acy27 mutant revealed that it contains a 78-bp insertion that apparently resulted from the duplication of adjoining sequences in exon 3 (data not shown). To determine whether the acy32 deletion (referred to as the ac29-3 allele) confers a more stringent acetate-requiring phenotype than the ac29-2 nonsense mutation, it was first necessary to create a haploid strain for the NIC7 ac29-3 mt+ chromosome. Such an ac29-3 strain was constructed by crossing the acy32 diploid to a green nic7 mt+ strain and identifying a prototrophic yellow mt+ strain among the progeny. DNA gel blot analysis (Figure 2B) demonstrated that the strain carried only the ac29-3 chromosome and failed to hybridize with the acC probe predicted to lie in the deleted region (Figures 1 and 2C). The haploid ac29-3 strain, like ac29-2, is not strictly acetate requiring. It grows normally on Tris-acetate phosphate (TAP) medium under high light. However, its growth on minimal medium or on TAP medium under low light is retarded significantly (Figure 5). It grows more slowly on high-salt medium (HSM) plates than ac29-2 (data not shown). Transformation of ac29-3 was performed with linearized pAc5.8. Only 2 x 106 cells were transformed, and they were spread onto 18 HSM plates. Several green sectors were isolated, and ultimately, 11 independent green transformants were isolated after subcloning. These results indicate that the yellow color of ac29-3 is caused by the deletion in the AC29 locus. This mutant was used for further studies.
The N-terminal part of the Alb3.1 protein of Chlamydomonas is rich in Ser, Thr, and Arg, a typical feature of chloroplast transit peptides, and is predicted by the Chloro P program to contain a chloroplast transit sequence (Emanuelsson et al., 1999 ). This is consistent with the location of the Arabidopsis Alb3 protein in the chloroplast (Sundberg et al., 1997) and with the finding that the Alb3.1 protein of Chlamydomonas is associated with thylakoid membranes (see below). Besides its similarity to the Alb3 protein of Arabidopsis, two regions of the Chlamydomonas protein, residues 128 to 232 and 284 to 358, show significant sequence identity to the Oxa1p mitochondrial protein of Saccharomyces cerevisiae and to the inner membrane YidC protein of E. coli (Figure 3). The C-terminal region contains a long stretch of Ala residues and a short highly positively charged region of seven residues. Figure 3 also indicates the positions of the Alb3 residues of Chlamydomonas and Arabidopsis that correspond to the intron insertion sites. It can be seen that among the five introns of Chlamydomonas and the nine introns of Arabidopsis, only intron 2 is inserted at the same site in both proteins. This finding suggests that, in contrast to the other introns, the second intron of Alb3 was present before the divergence of plants and green algae during evolution.
Localization of Alb3.1 The drug-resistant transformants were screened for expression of the tagged protein by immunoblotting with an HA antibody. Among 15 transformants tested, two (T1 and T2) expressed the Alb3-HA protein to detectable levels (Figure 6A). As expected, the tagged Alb3.1 protein was found in the thylakoid membrane fraction (Figure 6B). The purity of the fractions was tested with antibodies against RbcL and PsaE.
To determine the amount of Alb3.1 mRNA in the wild type and the rescued ac-29 strain, total RNA was isolated and examined by RNA gel blot analysis. Because the amount of RNA was undetectable by this method, reverse transcriptase–mediated (RT) PCR was performed using appropriate Alb3.1-specific oligonucleotides (see Methods). A fragment of the expected size was present in similar amounts in the wild-type and rescued strains and was absent, as expected, in the ac-29 mutant (Figure 6C, lanes 2 to 4). However, because the RT-PCR method used is semiquantitative, we were not able to measure accurately the relative levels of expression in the two strains.
The Alb3 Protein Is Required for the Normal Accumulation of LHC
To study the role of the Alb3.1 protein in the biogenesis of the photosynthetic apparatus, the levels of the different photosynthetic complexes in the thylakoid membranes of the wild type and the ac29-3 mutant were determined. Figure 7 shows the results obtained by immunoblotting of thylakoid membrane extracts and reacting the blots with antisera raised against specific subunits of PSI (PsaE), PSII (D1), the ATP synthase complex (  ), and the cytochrome b6f complex (Cytf). In all of these cases, no significant difference was observed between mutant and wild-type cells grown in the light, with the exception of the level of PSII, which was reduced twofold in ac29-3 cells.
The effect of the ac29-3 mutation on PSII was more pronounced in dark-grown cells, in which this complex was reduced nearly 10-fold. The major effect of the loss of Alb3.1 was the strong reduction of the LHC polypeptides (Figure 7). Quantitative immunoblot analysis with the LHCI-specific P14.1 and the LHCII-specific P11 antibodies indicated that LHCI accumulates to <5% and that LHCII accumulates to 5 to 10% of wild-type levels in light-grown mutant cells, in agreement with the chlorophyll measurements. This observed decrease of LHC is compatible with the chlorophyll remaining in ac29-3, 30% relative to the wild type.
The chlorophyll antennae from PSII and PSI of Chlamydomonas have been estimated at 320 and 290 chlorophyll molecules, respectively (Polle et al., 2000
Alb3.1-HA Is Associated with Two Complexes
The first peak of Alb3.1, with a size in the 60- to 80-kD range, cofractionates with LHCII, and the second peak corresponds to a high molecular mass complex, in the 600- to 700-kD range, that is slightly larger than the PSI complex. However, because the multiple forms of these complexes have been observed with the HA-tagged Alb3.1 protein, and because the tag could alter the properties of Alb3.1, it remains to be determined whether the untagged protein also is associated with two complexes.
Chlamydomonas Contains Two Alb3-Related Genes Alb3.2 displays 53% sequence identity and 71% sequence similarity to the Alb3 protein of Arabidopsis. The relatedness between Alb3.1 and the Arabidopsis protein is slightly smaller (46% identity and 65% similarity). These three Alb3 proteins have significantly more sequence similarity between themselves than to the yeast mitochondrial Oxa1p protein (19% sequence identity and 38% sequence similarity to Alb3.1, 18% sequence identity and 37% sequence similarity to both Alb3.2 and Alb3 of Arabidopsis). The relatedness to the bacterial YidC protein also is similar for Alb3.1 (27% sequence identity and 47% sequence similarity), Alb3.2 (31% sequence identity and 50% sequence similarity), and Alb3 of Arabidopsis (30% sequence identity and 48% sequence similarity).
The relationship between these different proteins is illustrated by the dendrogram at the bottom of Figure 3, which shows that Alb3.2 is related more closely to Alb3 of Arabidopsis than to Alb3.1. The Alb3 proteins of Chlamydomonas and Arabidopsis contain very similar hydrophobicity profiles, with five common putative transmembrane domains (data not shown). However, Alb3.1 appears to contain an additional transmembrane domain in its C-terminal region (data not shown). The Alb3.2 protein is likely to be a chloroplast protein, based on the prediction of the ChloroP program (Emanuelsson et al., 1999 In spite of their similar structure, it is likely that the functions of Alb3.1 and Alb3.2 differ significantly, because the Alb3.1-deficient ac29-3 mutant is affected drastically in the accumulation of LHCI and LHCII. However, because small amounts of LHCII still are present in this mutant, it is possible that Alb3.2 also plays some role in the assembly of this complex. Besides Alb3, a BLAST search for Alb3 homologs in Arabidopsis revealed another gene encoding a protein, named atAlb3.2, of 1013 residues that is related to Alb3 in its C-terminal half (45% identity and 67% similarity). Based on its N-terminal sequence, the atAlb3.2 protein is not predicted to be localized within the chloroplast, although the phlyogenetic tree indicates that its C-terminal part is closely related to Alb3 (Figure 3).
The AC29 Locus Encodes Alb3.1 In this study, we have shown that the AC29 locus encodes the Alb3.1 protein. This assignment is based on the physical characterization of several ac29 mutants with insertions and deletions at the AC29 locus, on the molecular characterization of pseudorevertants of the ac29-2 nonsense mutation that restore the open reading frame of Alb3.1, and on the rescue of the mutant phenotype by the transformation of ac29 mutants with the genomic Alb3.1 clone or with its cDNA.
The Alb3.1 protein shows a sequence identity of 42% to the Arabidopsis Alb3 protein. Furthermore, two regions of the protein display significant sequence identity to mitochondrial Oxa1p of yeast and the YidC inner membrane protein of E. coli. These proteins appear to be evolutionarily conserved mediators of membrane protein assembly in chloroplasts, mitochondria, and bacteria (Luirink et al., 2001
Inactivation of Alb3.1 in Chlamydomonas
Together, these results agree with the idea derived from in vitro experiments that Alb3 is connected closely to the SRP pathway (Woolhead et al., 2001
Role of Alb3 in LHC Assembly
The major portion of Alb3.1 is contained within a high molecular mass (600 to 700 kD) complex that is slightly larger than PSI. The current accepted model is that LHCP bound to chloroplast SRP in a soluble transit complex represents the LHCP targeted to the thylakoid membrane. LHCP integration requires the SRP receptor homolog cpFtsY, which partitions between stroma and thylakoids (Tu et al., 1999 Although our data support the view that Alb3.1 is part of a translocon system that is used specifically to integrate LHCII and LHCI into thylakoid membranes, as much as 10% of LHCII still accumulates in the absence of Alb3.1. Thus, an alternative pathway must exist for LHC integration into the thylakoid membrane. In this respect, it is interesting to note the existence of Alb3.2, a homolog of Alb3.1 that belongs clearly to the Alb3 family rather than to the Oxa1p group (Figure 3). Although this Alb3 homolog has not yet been characterized fully, it is possible that it also plays a role in LHC assembly and that it is partially redundant with Alb3.1. This could explain why LHC accumulation is not blocked fully in the absence of Alb3.1. However, it is clear that the functions of these two Alb3 homologs differ considerably. The ac29-3 mutant still is able to grow photoautotrophically, although at a much reduced rate.
A similar mutant with a disruption of the Alb3 gene has been described in Arabidopsis with a phenotype that is significantly more severe (Sundberg et al., 1997
Strains and Growth Conditions Wild-type and mutant strains of Chlamydomonas reinhardtii were maintained on solid Tris-acetate phosphate (TAP) medium supplemented with 4 µg/mL nicotinamide or 100 µg/mL Arg as needed. Sueoka's high-salt medium was used whenever an acetate-deficient medium was required (Harris, 1989 ). Crosses were performed using standard protocols (Harris, 1989). Transformations were performed using the glass beads/vortex protocol (Kindle, 1990).
DNA Gel Blot Analysis
cDNA Cloning
DNA Sequencing The segment of the ac29-2 allele from strain CC-44 that carries the BgIII-XbaI polymorphism was isolated as follows. PCR was performed on 25 ng of genomic DNA from the ac29-2 mutant using Vent DNA polymerase (New England Biolabs, Beverly, MA) primers 1 and 6 under the following conditions: 30 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min. The resulting fragment was gel purified and ligated into HincII-digested pUC118 and sequenced. Three potential revertants of ac29-2 were analyzed. Revertant 1 was a culture of CC-2663 received from the Chlamydomonas Genetics Center (Durham, NC) that was green, rather than the expected yellow, at the time of receipt. Revertant 2 was a culture of ac29-2 that has been in use in our laboratory for many years and had acquired a green color. Revertant 3 was a green colony isolated during an attempt to transform ac29-2 with a wild-type Alb3-1 genomic clone; it appeared not to contain any transforming DNA and therefore was assumed to be a revertant.
All three revertants, when examined by genomic DNA gel blot analysis, had retained the BglII polymorphism of the ac29-2 mutation but had lost the XbaI polymorphism. A PCR product was cloned from each strain (twice in the case of revertant 1) by the same protocol used for ac29-2 (except that primers 1 and 5 were used, with an annealing temperature of 66°C), and a segment of
The insertion in mutant acy27 was characterized as follows. PCR amplification was performed using 25 ng of acy27 genomic DNA and primers 1 and 5 under the following conditions: 30 cycles of 94°C for 1 min, 68°C for 1 min, and 72°C for 1 min. This resulted in the formation of two products, one of 558 bp (from the ac29-2 allele on the mt- chromosome) and one of 636 bp (from the new ac29 allele on the mt+ chromosome). The larger fragment was gel purified and ligated into HincII-digested pUC118, and the sequence of
Primers
Generation of New Mutants
The diploid strain was plated onto TAP at low density (
Transformation
Protein Extracts and Immunoblotting
Isolation and Characterization of the Alb3.2 cDNA The resulting PCR fragment was A-tailed (elongated with dATP; 2 mM dATP and 1 unit of TaqPol) for cloning into pDRIVE (Qiagen, Valencia, CA). The cloned fragment was verified by sequencing. A 380-bp EcoRI fragment from this plasmid was used as a probe to screen a cDNA library. Screening of 800,000 plaque forming units resulted in four positive clones containing inserts of 1.5, 1.8, 2.1, and 2.1 kb. Inserts were cut out with EcoRI, cloned into pBluescript KS-, and sequenced.
Procedures for the preparation of recombinant plasmids and DNA sequencing were performed as described by Sambrook et al. (1989)
RNA Analysis Five micrograms of RNA from the wild type was reverse transcribed with 50 units of Superscript reverse transcriptase, 0.5 µg of oligo(dT), 0.5 mM deoxynucleotide triphosphate, 5 mM MgCl2, 10 mM DTT, 20 mM Tris-HCl, pH 8.4, and 50 mM KCl for 50 min at 50°C in a total reaction volume of 50 µL. Three micrograms were used for PCR with Taq polymerase under the following conditions: 94°C for 4 min, then 30 cycles of 94°C for 1 min, 54°C for 45 s, and 72°C for 30 s, followed by 72°C for 4 min.
Sequence Analysis Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
We thank M. Goldschmidt-Clermont for helpful comments, L. Small for technical assistance, J. Pawlowski for help with the phylogenetic analysis, and N. Roggli for preparing the figures. This work was supported by a fellowship from EMBO and the Danish Natural Science Foundation to H.N., by National Science Foundation Grant MCB-9904667 to P.F., and by Grant 3100-050895.97 from the Swiss National Fund to J.-D.R.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003442.
1 These authors contributed equally to this work. Received March 27, 2002; accepted May 23, 2002.
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Abstract
INTRODUCTION
-subunit gene (Schloss, 1990
260 residues, is significantly more conserved (46% identity and 65% similarity). 
EMBL3 chromosome walk from strain CC-621 (mt-) (Ferris and Goodenough, 1994
C). This is within the second intron and represents a strain difference rather than a mutational change. 
