Porphobilinogen deaminase is encoded by a single gene in Arabidopsis thaliana and is targeted to the chloroplasts

Abstract

Porphobilinogen deaminase (PBG deaminase) is an early enzyme of the pathway for chlorophyll and heme synthesis. Using degenerate oligonucleotide primers, based on amino acid sequence data for purified PBG deaminase from pea, a fragment was amplified from Arabidopsis genomic DNA by PCR, and then used to isolate both a cDNA and a genomic clone for PBG deaminase from Arabidopsis. The cDNA, shown to be full-length by primer extension, encodes a precursor protein of 382 residues, which can be imported into isolated chloroplasts and processed to the mature size. The genomic clone encodes an identical sequence to the cDNA, except for the presence of four introns within the coding region of the mature protein, and 1.7 kb of upstream sequence. There is no obvious TATA box within 50 bp of the transcription start. Southern blot analysis suggests that PBG deaminase is encoded by a single gene in the Arabidopsis genome, and RNase protection experiments demonstrated that this gene is expressed in both leaves and roots. These results support the conclusion that there is only one form of PBG deaminase in all plant cells, which is located in the plastid.

A Single Precursor Protein for Ferrochelatase-I fromArabidopsis Is Imported in Vitro into Both Chloroplasts and Mitochondria

“…(14); lane 2, incubation with mitochondria followed by treatment with Proteinase K; lane 3, incubation with mitochondria (total import reaction); lane 4, incubation with mitochondria, followed by treatment with Proteinase K in the presence of Triton X-100; lane 5, incubation with mitochondria pretreated with valinomycin; lane 6, incubation of translation products with mitochondrial matrix extract (equivalent to 500 g of protein). The porphobilinogen precursor is 45 kDa, whereas the mature protein is 40 kDa (14). The extra band of 43 kDa seen in lane 3 is therefore not the mature protein and may arise from the action of nonspecific proteases in the mitochondrial preparation.…”

“…The precursor (45 kDa) is found associated with the mitochondria, together with another slightly smaller protein of 43 kDa (lane 3). This is not the mature protein, which is 40 kDa (14). Neither of the bands in the total import reaction is protease-resistant (lane 2), indicating that they are on the outside of the mitochondrial membrane, completely accessible to the protease.…”

“…The protein assay kit was from Bio-Rad. Plasmid pGef3 contained full-length ferrochelatase-I cDNA from Arabidopsis (13), and plasmid pAc222 contained the full-length cDNA for porphobilinogen deaminase from Arabidopsis (14). Plasmid pLHCP II contained full-length lightharvesting chlorophyll protein cDNA from pea (15) (a gift from Professor J. C. Gray, Dept.…”

“…We therefore used a precursor for another tetrapyrrole biosynthesis enzyme, porphobilinogen deaminase, which had previously been shown to be imported into pea chloroplasts (14) and which had been determined to be located exclusively within the plastids (30). Fig.…”

Siroheme Biosynthesis in Higher Plants

“…Import into Isolated Pea Organelles-For in vitro organelle import studies, the EcoRI insert from pUPM1 was subcloned into the pGEM3zf vector downstream of the SP6 promoter. This construct was transcribed and translated in the wheat germ system in the presence of [ 35 S]Met as described by Lim et al (37). Chloroplasts for the import assay were isolated from 7-to 10-day-old pea plants (Pisum sativum, variety Feltham First) grown in soil at 25°C under a 16-h light:8-h dark cycle, by a procedure based on that of Bartlett et al (38), except that a two-stepped (40 and 85%) discontinuous Percoll gradient was used in place of a continuous one.…”