SPECIFIC LABELING OF GLYCOPROTEINS IN YEAST PLASMA-MEMBRANE WITH CONCANAVALIN-A

  • 1 January 1981
    • journal article
    • research article
    • Vol. 25  (1) , 58-65
Abstract
The intramembranous particles of Saccharomyces cerevisiae plasma membrane form paracrystalline arrays or are randomly distributed as seen by freeze-fracture electron microscopy. Protoplasts with randomly distributed particles and with paracrystalline arrays were isolated and subsequently labeled with 3H-Con A (concanavalin A), Con A and ferritin-Con A. The distribution of the Con A or the ferritin-Con A molecules on deep-etched exoplasmic surfaces strongly resembled the distribution of the intramembranous particles. The influence upon labeling of buffer ionic strength was investigated. Binding assays with 3H-Con A and freeze-etch electron microscopy demonstrated that the amount of non-specifically bound lectin molecules decreases by increasing buffer ionic strength. Only partial removal of Con A molecules was achieved by adding various concentrations of the specific sugar Methyl-.alpha.-D-Mannoside (.alpha.MM) to labeled protoplasts. By analytical ultracentrifugation it was found that .alpha.MM also promotes the formation of Con A dimers. Fixed protoplasts were treated with detergents and 2-chloroethanol at various concentrations and subsequently labeled with 3H-Con A or ferritin-Con A. The amount of Con A bound to extracted cells did not decrease but ultrastructural changes of the deep-etched surfaces were observed. These data indicate that only the glycoproteins are labeled with Con A and they seem to be associated with the intramembranous particles. Each intramembranous particle seems to bind 36 to 44 Con A molecules and therefore the glycoproteins seem to possess very long sugar chains. This further supports the hypothesis that the intramembranous particles are associated with the membrane-bound invertase.

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