Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis

Abstract

A cDNA expression library was constructed from light‐grown Euglena gracilis poly(A)‐rich RNA in λgt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full‐length clone was isolated, which encoded not only the entire mature protein (Mr 36 927), but also an N‐terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51 744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)‐rich RNA. The mature protein is 60–70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N‐terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear‐encoded Euglena protein.

Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction

“…Initial attempts to isolate cDNAs encoding the pea PBG deaminase from Xgtl 1 expression libraries prepared from leaf poly(A) + RNA by direct hybridization with heterologous DNA probes encoding the enzyme from Euglena (Sharif et al, 1989), £. coli (Alefounder et al, 1988), and humans (Raich et al, 1986) were not successful.…”

“…This is perhaps not surprising given the nucleotide sequence identity between the different cDNAs, which is less than 50%. More unexpected, however, was a similar inability to successfully isolate phage encoding the PBG deaminase by immunoscreening of these Xgtll expression libraries using antiserum prepared against purified PBG deaminase from either Euglena or pea chloroplasts (Sharif et al, 1989;Spano and Timko, 1991). As a consequence, we adopted an alternative approach, in which primary protein sequence information was used to design a set of degenerate oligonucleotides that were subsequently used in Taql polymerase-catalyzed PCR amplifications from pea genomic DNA and cDNA.…”

“…Of the nine peptide sequences obtained, six (peptides a, c, d, e, g, and h) could be aligned with some confidence with the sequences of PBG deaminases from other organisms, whereas the location of the remaining three peptides (b, f, and j) were ambiguous and were presumed to be from more variable regions, such as the carboxy terminus of the protein (Sharif et al, 1989). Subsequent molecular cloning and analy- sis (discussed below) resolved the location of a11 peptide sequences and confirmed both the identity of the isolated cDNA and nuclear fragments as encoding the pea PBG deaminase and the deduced amino acid sequences of these clones.…”

“…Cloned cDNAs and nuclear genes encoding PBG deaminase have now been analyzed from severa1 mammalian cells (e.g. Raich et al, 1986;Beaumont et al, 1987;Chretien et al, 1988;Stubnicer et al, 1988), yeast (Gellefors et al, 1986;Keng et al, 1992), bacteria (Thomas and Jordan, 1986;Hansson et al, 1991), and one photosynthetic organism, Euglena gracilis (Sharif et al, 1989).…”

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

“…Euglena and dinoflagellate chloroplasts have three membranes, whereas diatom chloroplasts have four (Gibbs, 1981). Cytoplasmically synthesized chloroplast protein precursors of Euglena (Sharif et al, 1989;Chan et al, 1990;Kishore et al, 1993), dinoflagellates (Norris and Miller, 1994), and diatoms (Bhaya and Grossman, 1991;Kroth-Pancic, 1995) contain an ER-targeting signal peptide domain at the presequence N terminus. The Euglena precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II (pLHCPII) (Kishore et al, 1993) and the Phaeodactylum fucoxanthin chlorophyll a/c binding protein precursor (Bhaya and Grossman, 1991) are inserted cotranslationally into canine microsomes and the signal peptide is cleaved, suggesting that in organisms whose chloroplasts are surrounded by more than two membranes, chloroplast proteins are transported cotranslationally into the ER before chloroplast localization.…”