Purification and cloning of two high molecular mass isoforms of peanut seed oleosin encoded by cDNAs of equal sizes.
Oleosins are small plant proteins characterized by a long hydrophobic core flanked by amphipathic N- and C-terminal domains, which act as emulsifiers for the storage of lipids in seeds. They have been sequenced in a number of oilseeds important for the food industry but not in peanuts. We purified the major isoform of peanut oleosin by preparative electrophoresis with continuous elution, in sufficient amounts to raise specific antibodies, perform circular dichroism and N-sequence tryptic fragments. The structure of the purified oleosin was dominated by alpha-helix that may be assigned to the SDS-resistant central hydrophobic stretch. A two-step RT-PCR strategy was developed to determine the cDNA sequence of this oleosin. Two cDNA variants of equal sizes encoding for isoforms of 176 amino acids each were identified. The isoforms differed by seven amino acids mainly located in the N- and C-terminal domains. The corresponding mRNAs were estimated at 0.9 kb by Northern blot and were transcribed from genes without introns. Immunoprecipitation of the in vitro-translated peanut oleosin labeled with [14C]leucine or [35S]methionine produced the full-length protein (17 kDa) and a 6-kDa peptide corresponding to the N/C-terminal domains. This peptide was able to form SDS-PAGE stable oligomers by interacting with the full-length protein. A similar peptide was released after [125I]iodination of the purified oleosin that generated intermediate-sized oligomers also visible by Western blot on a crude oleosin extract. Oligomers reflect the natural ability of oleosins to strongly interact with each other via not only their central domains but also their N- and C-terminal domains.
Pons
Laurent
L
Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, Inserm EMI 0014, Faculté de Médecine, Université Henri Poincaré Nancy 1, BP 184, 54505 Vandaeuvre-lès-Nancy cedex, France.
Chéry
Céline
C
Mrabet
Nadir
N
Schohn
Hervé
H
Lapicque
Françoise
F
Guéant
Jean-Louis
JL
D000595:Amino Acid Sequence; D010367:Arachis / Q000235:genetics*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins