title: Protein targeting across the three membranes of the Euglena chloroplast envelope.
abstract: A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes. Preparation of functional chloroplasts with intact envelope membranes has been optimized. Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62%. Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors. The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa). A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported. Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope. Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size. However, this was not imported into Euglena chloroplasts, although again it bound to them. These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence. The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.
authors.affiliation: Department of Plant Sciences, University of Cambridge, United Kingdom.
authors.forename: L S
authors.initials: LS
authors.lastname: Shashidhara
authors.forename: S H
authors.initials: SH
authors.lastname: Lim
authors.forename: J B
authors.initials: JB
authors.lastname: Shackleton
authors.forename: C
authors.initials: C
authors.lastname: Robinson
authors.forename: A G
authors.initials: AG
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA; D004792:Enzyme Precursors / Q000378:metabolism; D005055:Euglena / Q000378:metabolism*; D011163:Hydroxymethylbilane Synthase / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals
chemical_list: D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D004247:DNA; D011163:Hydroxymethylbilane Synthase