ABSTRACT
Using a novel cyanelle isolation procedure we showed that pre-ferredoxin-NADP+-oxidoreductase (pre-FNR) from C. paradoxa is translocated in vitro across the peptidoglycan-containing cyanelle envelope. Efficient import was also observed in a heterologous system with pea chloroplasts as the recipient organelles. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semi-autonomous endosymbiotic ancestor.
Subject(s)
Chloroplasts/metabolism , Enzyme Precursors/metabolism , Eukaryota/enzymology , Ferredoxin-NADP Reductase/metabolism , Pisum sativum/metabolism , Plastids/enzymology , Cell Fractionation , Centrifugation , Enzyme Precursors/biosynthesis , Enzyme Precursors/isolation & purification , Eukaryota/ultrastructure , Ferredoxin-NADP Reductase/biosynthesis , Ferredoxin-NADP Reductase/isolation & purification , Molecular Weight , Peptidoglycan/metabolism , Plastids/ultrastructure , Transcription, GeneticABSTRACT
The petFI gene encoding ferredoxin I was localized in the large single copy region of cyanelle DNA by heterologous hybridization. Sequence analysis revealed an ORF of 99 amino acids (including the N-terminal processed methionine) at a position 477 bp from the 3' end of tufA but on the opposite strand. The 25 amino-terminal residues well corresponded to partial sequences obtained with purified cyanelle ferredoxin. The assignment of yet another gene that is not found on the genomes of chlorophyll b-type plastids to cyanelle DNA again corroborates the special position of cyanelles serving as a model for plastid evolution from endocytobiotic cyanobacteria.
Subject(s)
Eukaryota/genetics , Ferredoxins/genetics , Genes, Plant , Organelles , Amino Acid Sequence , Base Sequence , Blotting, Northern , Chromosome Mapping , Molecular Sequence Data , Operon , RNA, Messenger/geneticsABSTRACT
Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interference from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.
Subject(s)
Membranes, Artificial , Monosaccharide Transport Proteins/chemistry , Biotin/chemistry , Cell Compartmentation , Electrochemistry , Electrodes , Erythrocyte Membrane/chemistry , Glucose/analysis , Glucose Transporter Type 1 , Humans , Membrane Lipids/analysis , Microscopy, Atomic Force , Spectrophotometry, InfraredABSTRACT
rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the "S10" operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.
Subject(s)
DNA, Ribosomal/genetics , Eukaryota/genetics , Organelles/metabolism , Plant Proteins/genetics , Ribosomal Proteins/genetics , Amino Acid Sequence , Archaea/genetics , Bacteria/genetics , Base Sequence , Extrachromosomal Inheritance , Gene Expression Regulation , Molecular Sequence Data , Operon , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR. The 1.4 kb transcript was derived from a single-copy gene. The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively. The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs. Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C. paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences. The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides.