Cytoplasmic N-terminal protein acetylation is required for efficient photosynthesis in Arabidopsis

Abstract

The Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II. In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes. DNA array-based mRNA analysis indicated that extraplastid functions also are altered. The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p. In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC). The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein. This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast. We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts.

Figure 1.

Phenotypes of pam21 and Wild-Type Plants. (A) Four-week-old plants grown in the greenhouse. WT, wild type. (B) Growth kinetics of pam21 (n = 100) compared with wild-type (n = 100) plants. Leaf area was measured in the period from 5 to 20 days after germination. Error bars indicate standard deviations.

Figure 2.

Protein Composition of Thylakoid Membranes. (A) Thylakoid proteins from identical amounts (fresh weight) of wild-type (WT) and pam21 leaves were fractionated first by electrophoresis on a nondenaturing lithium dodecyl sulfate polyacrylamide (LDS-PA) gel and then on a denaturing SDS-polyacrylamide (SDS-PA) gel. Positions of wild-type thylakoid proteins identified previously by protein gel blot analyses with appropriate antibodies are indicated: 1, α- and β-subunits of the ATPase complex; 2, D1-D2 dimer; 3, CP47; 4, CP43; 5, oxygen-evolving complex; 6, LHCII monomer; 7, LHCII trimer; 8, PSI-D; 9, PSI-F; 10, PSI-C; 11, PSI-H. (B) Thylakoid proteins from identical amounts (fresh weight) of wild-type and pam21 leaves fractionated by one-dimensional PAGE. Decreasing levels of wild-type thylakoid proteins were loaded in the lanes marked 0.8×WT, 0.6×WT, 0.4×WT, and 0.2×WT. Three replicate filters were probed with antibodies raised against PSI-A/B, D1, and LHCII.

Figure 3.

mRNA Expression in Chloroplasts. The numbers at right indicate RNA sizes as estimated by coelectrophoresis with denatured EcoRI-HindIII fragments of λDNA. Aliquots (30 μg) of total RNA from the wild type (WT) and pam21 were size-fractionated by agarose gel electrophoresis, transferred to a nitrocellulose filter, and probed with cDNA fragments specific for psbA, psbB, psbC, psaA, psaB, atpA, and atpB.

Figure 4.

In Vivo Synthesis of Thylakoid Proteins and Polysome Accumulation. (A) ³⁵S-Met was applied for 10 min (top gels) or 30 min (bottom gels) to 4-week-old leaves under illumination. Thylakoid proteins from 100-mg portions of wild-type and pam21 leaves were separated by blue-native PAGE in the first dimension and by SDS-PAGE in the second dimension, electroblotted onto a nylon membrane, and analyzed by fluorography. Results from one of three independent experiments are shown, and the identities of the labeled bands are indicated at right according to Plucken et al. (2002). (B) Total extracts from pam21 and wild-type leaves were fractionated on sucrose gradients. Fourteen fractions of equal volume were collected from the top to the bottom of the sucrose gradients. An equal proportion of the RNA purified from each fraction was analyzed by gel blot hybridization. Transcripts of psbA, psbB, and psbC were detected with gene-specific probes. Results from one of three independent experiments are shown.

Figure 5.

T-DNA Tagging and mRNA Expression of the At2g38130 Gene. (A) At2g38130 was disrupted by an insertion of the 5.8-kb AC106 T-DNA in the third intron. The T-DNA insertion is not drawn to scale. Lowercase letters indicate plant DNA sequences flanking the T-DNA insertion. LB, left border; RB, right border. (B) RNA gel blot analysis of the At2g38130 transcript in pam21 and wild-type (WT) leaves. Samples (30 μg) of total RNA were analyzed using At2g38130 full-length cDNA as a probe. To control for RNA loading, the blot was reprobed with a cDNA fragment derived from the APT1 gene, which is expressed at a low level in all tested tissues of Arabidopsis (Moffatt et al., 1994). (C) Detection of At2g38130 transcripts in different plant organs by quantitative reverse transcriptase–mediated PCR. The analysis on 4.5% polyacrylamide gels was performed with ³³P-labeled quantitative reverse transcriptase–mediated PCR products from roots, leaves, flowers, and siliques obtained after PCR for 20 cycles with At2g38130-specific primers (atmak3-137s and ³³P-labeled primer atmak3-1494as) and control primers for the APT1 gene (Moffatt et al., 1994). (D) The same analysis as in (C) was performed for leaves at different developmental stages.

Figure 6.

Comparison of Mak3p-Like Sequences from Eukaryotes. The amino acid sequence of the At2g38130 protein was compared with homologous sequences from S. cerevisiae (Mak3p), S. pombe (7492223), D. melanogaster (7511971 and 7292084), C. elegans (17557298), and human (17476873). Black boxes indicate strictly conserved amino acids, and gray boxes indicate closely related amino acids. Plus signs refer to positions of point mutations that abolish Mak3p function (Tercero et al., 1992). Conserved sequence stretches (motifs A to D) that are typical of NATs are indicated according to Neuwald and Landsman (1997).

Figure 7.

Intracellular Protein Localization. (A) Fluorescence field of mesophyll tobacco protoplasts (SR1) transfected with the At2g38130-dsRED fusion protein. (B) Bright-field view of the same section as in (A). Bar = 10 μm.

Figure 8.

At2g38130 Complements the Yeast mak3 Mutation. (A) The yeast mutant mak3 (Sommer and Wickner, 1982) was transformed with the Arabidopsis At2g38130 cDNA (At2g38130) and, as controls, with the S. cerevisiae MAK3 cDNA (ScMAK3) and with the empty vector alone (vector control). After cytoduction (Ridley et al., 1984), the three yeast strains were tested for their ability to propagate L-A-HNB M₁ dsRNA virus particles. Both mak3 ScMAK3 and mak3 At2g38130 strains were able to propagate the virus, as detected by the presence of the killing zones surrounding the yeast colonies. (B) The strains mak3 ScMAK3 (1), mak3 At2g38130 (2), and mak3 plus empty vector (3) were tested for their ability to grow on nonfermentable carbon sources (YPG) at 37°C (top half). As a control, strains were propagated on full medium (YPD; bottom half).

Figure 9.

Comparison of Yeast Mak10p and Mak31p Sequences with Orthologous Sequences from Arabidopsis. (A) Yeast Mak10p. (B) Yeast Mak31p. The D and F regions are motifs with similarity to domains of T cell receptor α-subunits (Lee and Wickner, 1992). Plus signs indicate amino acids conserved between Mak10p and T cell receptor α-subunits (A) and between Mak31p and Sm-like proteins (B). The asterisk in (B) indicates position 107 of the Sm-like protein consensus sequence according to Séraphin (1995). Black boxes indicate strictly conserved amino acids, and gray boxes indicate closely related amino acids.

Figure 10.

Yeast Two-Hybrid Analysis of Interactions between AtMAK3 and Candidate MAK10 and MAK31 Proteins from Arabidopsis. (A) The AtMAK3 protein fused to the GAL4 binding domain (AtMAK3^BD) was tested for interaction with At2g11000 fused to the GAL4 activation domain (At2g11000^AD). (B) The At3g11500 protein fused to the GAL4 binding domain (At3g11500^BD) was tested for interaction with At2g11000 fused to the GAL4 activation domain (At2g11000^AD). (C) The AtMAK3 protein fused to the GAL4 binding domain (AtMAK3^BD) was tested for interaction with At3g11500 fused to the GAL4 activation domain (At3g11500^AD). To detect interactions, transformed yeast strains were grown on either permissive (1) or selective (2) medium or subjected to the LacZ assay (3). AD, empty vector containing the GAL4 activation domain; BD, empty vector containing the GAL4 binding domain.

Figure 11.

AtMAK3 Can Replace Mak3p, Mak10p, and Mak31p. (A) The mutant yeast strain mak10 (Sommer and Wickner, 1982) was transformed with the S. cerevisiae MAK10 cDNA (ScMAK10), with the Arabidopsis At2g11000 cDNA (At2g11000), or with the empty vector (vector control). After cytoduction (Ridley et al., 1984), the three yeast strains were tested for the ability to propagate the L-A-HNB M₁ dsRNA virus. Only ScMAK10 was able to propagate the virus, as detected by the presence of killing zones surrounding the yeast colonies. (B) The double mutant yeast strain mak3 mak10 was transformed with both ScMAK3 and ScMAK10 cDNAs (ScMAK3 ScMAK10), with both AtMAK3 and At2g11000 cDNAs (AtMAK3 At2g11000), with the AtMAK3 cDNA (AtMAK3), with the ScMAK3 cDNA (ScMAK3), with the At2g11000 cDNA (At2g11000), or with the empty vector (vector control). Only ScMAK3 ScMAK10, AtMAK3 At2g11000, and AtMAK3 were able to complement the double mutation, enabling propagation of the L-A-HNB M₁ dsRNA virus. (C) The mutant yeast strain mak10 was transformed with the S. cerevisiae MAK10 cDNA (ScMAK10), with the Arabidopsis AtMAK3 cDNA (AtMAK3), with the S. cerevisiae MAK3 cDNA (ScMAK3), or with the empty vector (vector control). ScMAK10 and AtMAK3 were able to complement the mutation. (D) The mutant yeast strain mak31 was transformed with the Arabidopsis AtMAK3 cDNA (AtMAK3), with the S. cerevisiae MAK3 cDNA (ScMAK3), or with the empty vector (vector control). As a control, an isogenic wild-type strain (WT) was used. Only AtMAK3 was able to complement the mutation.

Figure 12.

Effects of the atmak3-1 Mutation on the Accumulation of Nuclear Transcripts of Genes for Chloroplast Proteins. Total RNA isolated from wild-type and atmak3-1 mutant plants was used to probe a nylon filter DNA array carrying 3292 gene sequence tags. In atmak3-1, 777 of the 3292 genes tested were significantly differentially expressed compared with wild-type plants. The 577 differentially expressed chloroplast protein–coding genes in atmak3-1 were grouped into seven major functional categories. A complete list is available in the supplemental data online.