Identification of a signal peptide for oryzacystatin-I.
A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5' gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site. which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5' end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Womack
J S
JS
Department of Entomology, Plant Pathology and Weed Science, New Mexico State University, Las Cruces 88003, USA.
Randall
J
J
Kemp
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
D015891:Cystatins; D018744:DNA, Plant; D004274:DNA, Recombinant; D011498:Protein Precursors; D021382:Protein Sorting Signals