Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg(2+) added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[(14)C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[(14)C]mannose, [(14)C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [(14)C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [(14)C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [(14)C]sugar moiety was associated with several, but not all, constituent polypeptides.