Heat-induced synthesis and tunicamycin-sensitive secretion of the putative storage glycoprotein conglutin gamma from mature lupin seeds

SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60 degrees C for about 3 h was conglutin gamma, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin gamma was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin gamma. Synthesis and secretion took place only over a narrow temperature range, 57.5-62.5 degrees C, and in a short time interval, 135-180 min, of incubation of the seed. The amount of secreted conglutin gamma, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed. Secreted conglutin gamma contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin gamma synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin gamma remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry. Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin gamma with trypsin and pepsin revealed no difference between the two proteins. Lupin seeds were still viable after the treatment. However no similarities between conglutin gamma and heat-shock proteins were observed either in the amino acid sequence or other molecular features.