SUBCELLULAR-LOCALIZATION OF MANNOSYL TRANSFERASE AND GLYCOPROTEIN-BIOSYNTHESIS IN CASTOR BEAN ENDOSPERM

Differential and sucrose density graident centrifugation showed that the mannosyl transferase present in germinating castor bean [Ricinus] 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 Mg2+ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[14C]mannose demonstrated that most of the mannolipid formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which was previously incubated with GDP[14C]mannose, [14C]glycoprotein was associated with other cellular fractions in addition to the microsomes, particularly the glyoxysomes. The kinetics of radioactive labeling of these organelles suggest that [14C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated [14C]glycoprotein-labeled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [14C]sugar moiety was associated with several, but not all, constituent polypeptides.