Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

Abstract

A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on two specific nucleus-encoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation. We cloned the TCA1 gene, encoding a pioneer protein, and transformed appropriate mutant strains with tagged versions of MCA1 and TCA1. In transformed strains expressing decreasing amounts of MCA1 or TCA1, the concentration of these factors proved limiting for petA mRNA accumulation and cytochrome f translation, respectively. This observation suggests that in exponentially growing cells, the abundance of MCA1 sets the pool of petA transcripts, some of which are TCA1-selected for an assembly-dependent translation of cytochrome f. We show that MCA1 is a short-lived protein. Its abundance varies rapidly with physiological conditions that deeply affect expression of the petA gene in vivo, for instance in aging cultures or upon changes in nitrogen availability. We observed similar but more limited changes in the abundance of TCA1. We conclude that in conditions where de novo biogenesis of cytochrome b(6)f complexes is not required, a rapid drop in MCA1 exhausts the pool of petA transcripts, and the progressive loss of TCA1 further prevents translation of cytochrome f.

Fig. 1.

Tagged versions of TCA1 (A) or MCA1 (B). In both A and B, the first line displays a schematic representation of the structure of the gene with initiation and stop codons. Exons are shown as gray boxes. The two following lines represent the constructs used for complementation of tca1 (A) or mca1 (B) mutant strains. The position of the tags with respect to the coding sequence is shown. Length of sequences upstream of the initiation codon (A is taken as +1) is indicated, as well as introns when retained in the construct. PolyA tail indicates constructs derived from a cDNA clone. (A) E and A refer to restriction sites EcoRI and AccI, between which the flag tag was inserted in frame.

Fig. 2.

MCA1 and TCA1 are targeted to the chloroplast. Whole cell (Cells), chloroplast (Cp), and mitochondrial (Mt) fractions, purified from cell wall-deficient strains expressing MCA1-HA and TCA1-fl respectively. UDP-glucose pyrophosphorylase (UGPase), subunit β of mitochondrial ATP synthase (F1β), and GRPE provide controls for fraction purity.

Fig. 3.

MCA1 is rate-limiting for petA mRNA accumulation. (A) Accumulation of MCA1-HA, cyt. f, and GRPE (loading control) detected with specific antibodies in independent clones complemented with construct lgMCA1-HA. Accumulation of petA and CβLP2 (loading control) mRNAs in the same strains is shown below. Asterisks indicate the clones used for experiments in Figs. 5–7 (strains M-HA). (B) Relationship between MCA1-HA abundance (arbitrary units) and petA mRNA accumulation (reported to that in the wild type). The graph combines data obtained from clones transformed with construct shMCA1-HA (triangles) and from clones presented in A (squares). (C) Relationship between MCA1-HA (arbitrary units) and cyt. f accumulation (percent of the wild-type level) in strains from A.

Fig. 4.

TCA1 is rate-limiting for cyt. f accumulation. (A) Accumulation of TCA1-fl, cyt. f and GRPE (loading control) in independent clones transformed with construct shTCA1-fl. (B) Correlation between TCA1-fl abundance and cyt. f accumulation. Data are from clones from A (squares) and from clones transformed with lgTCA1-fl (triangles). Clones indicated by an asterisk were used for experiments on Figs. 5–7 (strains T-fl). (Inset) The recovery of petA mRNA in three representative complemented strains.

Fig. 5.

Stability of MCA1-HA and TCA1-fl. Accumulation of MCA1-HA (A), TCA1-HA (C), cyt. f in M-HA (A), wild-type (B), or T-fl (C) strains treated with cycloheximide for the indicated times. Accumulation of petA mRNA and 16S rRNA (loading control) in the same strains is also shown.

Fig. 6.

Accumulation of MCA1-HA and TCA1-fl decreases during aging of cultures. Wild-type, M-HA (A), or T-fl (B) strains were inoculated at a density of 0.5 × 10⁶ cells per ml and grown up to 5 days. Abundance of MCA1-HA (A), TCA1-fl (B), and cyt. f proteins and of petA and psbA transcripts were monitored at early exponential (EE) (1–2 × 10⁶ cells per ml), late exponential (LE) (4–5 × 10⁶ cells per ml) and stationary (St) (6–7 × 10⁶ cells per ml) phases of growth. GRPE and CβLP2 provide the respective loading controls.

Fig. 7.

MCA1-HA and TCA1-fl are reversibly down-regulated during nitrogen starvation. Accumulation of MCA1-HA (A), TCA1-fl (B), cyt. f, and F1β (loading control) in M-HA (A), wild-type (B), and T-fl (C) cells subjected to nitrogen starvation (−N) followed by repletion (+N) for the indicated times (in hours). Protein samples were loaded on an equal cell number basis. Accumulation of petA and 16S (as a loading control) transcripts in the same cells is also shown.