Loss of Albino3 leads to the specific depletion of the light-harvesting system

Abstract

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 b(6)f 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.

Figure 1.

Map of the AC29 Locus. The probes acB and acC are shown. The approximate location of the point mutations, insertions (wedges), and deletions (−) of the acy mutants are marked. The exons and introns of the Alb3 gene are indicated with black bars and lines, respectively. Untranslated regions are shown as open bars. Transcription proceeds from left to right. The primers used are indicated. Plasmids and phages used in transformations are shown at bottom. Rescue of the mutant phenotype is indicated by +. Restriction sites are indicated at top: Bg, BglII; Bs, BstEII; N, NsiI; R, EcoRI; Sc, SacI; Sf, SfiI; Sm, SmaI; Sp, SpeI; Xb, XbaI.

Figure 2.

DNA Gel Blot Analysis of ac29 Mutants. DNA was isolated from the diploid parent (nic7 ac29-2 mt⁻ pf14/ ++ mt⁺ +) and the diploid acy9, acy10, acy20, acy25, and acy32 mutant strains and from the haploid ac29-3 mutant containing the mt⁺ chromosome from acy32. The DNAs of the blots were digested with BglII (A) or SacI ([B] and [C]) and probed with the acB probe ([A] and [B]) or the acC probe (C).

Figure 3.

Sequence Comparison of the Alb3 Protein with YidC and Oxa1p. The protein sequences were aligned using ClustalW (Thompson et al., 1994) and Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Alb3.1 and Alb3.2 are from Chlamydomonas; atAlb3 (Sundberg et al., 1997) and atAlb3.2 are from Arabidopsis; YidC is from E. coli (Luirink et al., 2001); Oxa1p is from S. cerevisiae (Bonnefoy et al., 1994). The sites corresponding to the introns are indicated by open and closed wedges for Alb3.1 of Chlamydomonas and atAlb3 of Arabidopsis, respectively. In the phylogenetic tree at bottom, the numbers of amino acid substitutions per alignment site are indicated on the branches. The phylogenetic analysis was performed using the PHYLO_WIN program (Galtier and Gouy, 1996).

Figure 4.

Sequence Analysis of the Region of Alb3.1 Affected in the ac29-2 Mutant and Three Revertant Strains. Only the sequence surrounding the BglII site of ac29-2 is shown.

Figure 5.

Growth Patterns of the Wild Type (WT) and ac29-3. Five microliters from an exponentially growing culture was spotted on TAP or HSM (minimal) under different light conditions as indicated.

Figure 6.

Localization of the Alb3-HA Protein and Expression of the Alb3.1 and Alb3.2 Genes. (A) Immunoblot analysis of total cell extracts from the ac29-3 mutant, the wild type (WT), and two ac29-3 transformants (T1 and T2) rescued with the HA epitope–tagged Alb3 gene. The blot was decorated with HA antibody. (B) Cells from the T2 transformant were lysed and fractionated into a thylakoid membrane fraction (thyl) and a soluble fraction (sol). (C) At top, total RNA from wild-type, ac29-3, and T2 transformants was subjected to RT-PCR, and the products were fractionated by agarose gel electrophoresis using primers specific for Alb3.1 (lanes 2 to 4), Alb3.2 (lanes 6 to 8), and the G-protein β-subunit gene (Schloss, 1990) (control; lanes 13 to 15). The same reaction with the Alb3.1 primers without reverse transcriptase did not yield any signal (lanes 10 to 12). The same primers for Alb3.1 and Alb3.2 were used for PCR on wild-type genomic DNA (lanes 5 and 9, respectively). Lane 1 contained molecular mass markers (M). At bottom, the DNA fragments were blotted onto filters and hybridized with probes specific for Alb3.1 and Alb3.2 as indicated.

Figure 7.

The Abundance of LHCI and LHCII Is Reduced in the ac29-3 Mutant. (A) Thylakoid proteins from the wild type (WT), ac29-3, and ac29-3 rescued with a HA epitope–tagged Alb3 gene (T2) were separated by PAGE, immunoblotted, and decorated with antibodies against PSI (PsaE), PSII (D1), ATP synthase (α), cytochrome b₆f complex (Cytf), LHCI, and LHCII. (B) Dilutions of the protein extracts shown in (A) were used to estimate the amount of protein accumulated in ac29-3. D, darkness; L, light.

Figure 8.

The Alb3-HA Protein Is Associated with Two Complexes. Thylakoid membranes from the T2 transformant were solubilized with dodecyl maltoside and fractionated on a Suc density gradient. Fractions of 1 mL were collected, and 32-μL aliquots were used for PAGE and immunoblotting. The antibodies used are indicated at left. Antibodies specific for LHCII, PSII (PsbB), and PSI (PsaE) were used. Masses are indicated in kD and were determined by comparison with molecular mass standards provided with a molecular marker kit (Sigma). HA, HA-tagged Alb3 protein.