A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation.
We have examined the synthesis, posttranslational processing, and localization of soybean P34, a member of the papain superfamily. P34 has been identified as a constituent of oil storage organelles or oil bodies isolated from seed lysates and has been assumed to be one of the oil body proteins. Electron microscopic immunocytochemistry with a monoclonal antibody demonstrated that P34 is localized in the protein storage vacuoles but not in the oil bodies. Immunocytochemical observations of partially disrupted seed cells showed that the association of P34 with oil bodies appears to occur as a consequence of cell lysis. In vitro synthesis of P34 results in the formation of a 46-kDa polypeptide that increases to 47 kDa due to core glycosylation by canine microsomes. In vivo synthesis studies in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation, indicate that pro-P34 is 47 kDa. Since the cDNA sequence of prepro-P34 contains a single putative glycosylation site in the precursor domain, we conclude that P34, like a few other vacuolar proteins, is synthesized as a glycoprotein precursor. Pulse-chase experiments showed that the processing of pro-P34 to mature P34 occurs in a single step and that this posttranslational cleavage occurs on the carboxyl side of an Asn, which is typical of seed vacuolar proteins. Pro-P34 (47 kDa) is detected in immunoblots of maturing seeds. Analysis of RNA indicates that the P34 genes are expressed only during seed maturation and that the P34 mRNA is related to other thiol protease mRNAs detectable in other organs and plants. Unlike other seed thiol proteases that are synthesized only after seed germination, P34 accumulates during seed maturation.
Kalinski
A
A
Plant Molecular Biology Laboratory, United States Department of Agriculture, Beltsville, Maryland 20705.
Melroy
D L
DL
Dwivedi
R S
RS
Herman
E M
EM
D000485:Allergens; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012639:Seeds / Q000201:enzymology; D030262:Soybean Proteins; D013025:Glycine max / Q000196:embryology / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology* / Q000648:ultrastructure
D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; D030262:Soybean Proteins; D013438:Sulfhydryl Compounds; C075135:P34 protein, Glycine max; D004247:DNA; D003546:Cysteine Endopeptidases