Ubiquitin-Proteasome Dependent Regulation of the GOLDEN2-LIKE 1 Transcription Factor in Response to Plastid Signals.

Arabidopsis (Arabidopsis thaliana) GOLDEN2-LIKE (GLK) transcription factors promote chloroplast biogenesis by regulating the expression of photosynthesis-related genes. Arabidopsis GLK1 is also known to participate in retrograde signaling from chloroplasts to the nucleus. To elucidate the mechanism by which GLK1 is regulated in response to plastid signals, we biochemically characterized Arabidopsis GLK1 protein. Expression analysis of GLK1 protein indicated that GLK1 accumulates in aerial tissues. Both tissue-specific and Suc-dependent accumulation of GLK1 were regulated primarily at the transcriptional level. In contrast, norflurazon- or lincomycin-treated gun1-101 mutant expressing normal levels of GLK1 mRNA failed to accumulate GLK1 protein, suggesting that plastid signals directly regulate the accumulation of GLK1 protein in a GUN1-independent manner. Treatment of the glk1glk2 mutant expressing functional GFP-GLK1 with a proteasome inhibitor, MG-132, induced the accumulation of polyubiquitinated GFP-GLK1. Furthermore, the level of endogenous GLK1 in plants with damaged plastids was partially restored when those plants were treated with MG-132. Collectively, these data indicate that the ubiquitin-proteasome system participates in the degradation of Arabidopsis GLK1 in response to plastid signals.

Specific and Efficient Targeting of Cyanobacterial Bicarbonate Transporters to the Inner Envelope Membrane of Chloroplasts in Arabidopsis.

Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM) of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments.

The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM.

Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny.

Biogenesis of chloroplasts is essential for plant growth and development. A number of homozygous mutants lacking a chloroplast protein exhibit an albino phenotype. In general, it is challenging to grow albino Arabidopsis plants on soil until they set seeds. Homozygous albino mutants are usually obtained as progenies of heterozygous parents. Here, we describe a method of recovering seeds from the seedling lethal Arabidopsis mutant ppi2-2, which lacks the atToc159 protein import receptor at the outer envelope membrane of chloroplast. Using plastic containers, we were able to grow homozygous ppi2-2 plants until these set seed. Although the germination rate of the harvested seeds was relatively low, it was still sufficient to allow us to further analyze the ppi2-2 progeny. Using ppi2-2 homozygous seeds, we were able to analyze the role of plastid protein import in the light-regulated induction of nuclear genes. We propose that this method be applied to other seedling lethal Arabidopsis mutants to obtain homozygous seeds, helping us further investigate the roles of plastid proteins in plant growth and development.