Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C.
cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An approximately 2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor.
Shorrosh
B S
BS
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402.
Dixon
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007535:Isomerases / Q000235:genetics*; D046911:Macromolecular Substances; D000455:Medicago sativa / Q000201:enzymology / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D019704:Protein Disulfide-Isomerases; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014714:Vertebrates
D046911:Macromolecular Substances; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D012313:RNA; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases