Molecular characterisation of plant endoplasmic reticulum. Identification of protein disulfide-isomerase as the major reticuloplasmin.
Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase. Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein.
Coughlan
S J
SJ
Trait and Technology Development Department, Pioneer Hi-Bred Intemational, Johnston IA, USA.
Hastings
C
C
Winfrey
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017343:Genes, Plant; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D063245:Plant Development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010946:Plants, Medicinal; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D007535:Isomerases; D019704:Protein Disulfide-Isomerases