A Putative Mg Chelatase Subunit from Arabidopsis thaliana cv C24 (Sequence and Transcript Analysis of the Gene, Import of the Protein into Chloroplasts, and in Situ Localization of the Transcript and Protein

Abstract

We have isolated and sequenced a cDNA from Arabidopsis tbaliana cv C24 that encodes a putative Mg chelatase subunit. The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrbinum majus (olive) and also high similarity to bcbH, a bacterial gene involved i n the Mg chelatase reaction of bacteriochlorophyll biosynthesis. We suggest that this gene be called CHL H. Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, cb-42, and ferrochelatase. The CHL H transcript was observed to undergo a dramatic diurna1 variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily t o a minimum by the end of the light period; in contrast, transcripts for cb-42 and ferrochelatase remained constant. A model is proposed i n which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle. In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the cb-42 gene. The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay. lmmunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg2+. In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo.

Mechanism and regulation of Mg-chelatase

“…The major impediment was the lack of an in itro assay, which was only achieved 6 years ago [2]. Since that time our knowledge of Mg-chelatase has expanded dramatically to the extent that the enzyme has been cloned and sequenced from several photosynthetic species [3][4][5][6][7][8][9][10].…”

“…Mg-chelatase is readily solubilized from membranes in the absence of Mg [5,19,27]. The solubilized proteins have been separated into three fractions by pseudoaffinity chromatography and ultrafiltration [28], and all three fractions are necessary for Mg-chelatase activity.…”

“…These proteins range in size from 37 kDa for the chloroplast-encoded subunit I of Olisthodiscus luteus (a unicellular alga) to 46 kDa for the nuclear-encoded Arabidopsis thaliana (thale cress) subunit I counterpart which includes a Nterminal chloroplast transit peptide [5]. It is interesting that only in the cases of the non-chlorophyte algae are the subunit I sequences encoded by the chloroplast genome.…”

“…The remaining crude envelope and stromal proteins were highly active, indicating that Mg-chelatase was associated with the envelope membranes. Now, with the information that Mgchelatase is a soluble enzyme in the absence of Mg [5,19,27], these data can be reinterpreted : during lysis and removal of the thylakoids, Mg-chelatase would have been soluble : on the subsequent addition of Mg# + the enzyme would have been driven on to the envelope membranes.…”

“…This question may be settled by in itro import studies and immunogold labelling. Already the import of the H and I subunit into the stroma of Arabidopsis chloroplasts has been reported, although their definitive association with either the envelopes and thylakoids has not yet been shown [5].…”