Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm |
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Authors: | R B Mellor J M Lord |
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Institution: | (1) Postgraduate School of Biological Sciences, University of Bradford, BD7 IDP Bradford, U.K. |
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Abstract: | 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 Mg2+ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP14C]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 GDP14C]mannose, 14C]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 14C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, 14C]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 14C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER
endoplasmic reticulum
- TCA
trichloroacetic acid
- SDS
sodium dodecylsulphate
- GDP
guanosine diphosphate |
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Keywords: | Endoplasmic reticulum Glycoprotein Glyoxysomes Microsomes Ricinus |
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