Specific labeling of glycoproteins in yeast plasma membrane with concanavalin A.
The intramembranous particles of yeast Saccharomyces cereisiae 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, 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 means of 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. From our data it can be concluded that only the glycoproteins are labeled with Con A and they seem to be associated with the intramembranous particles [15]. 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.
D002462:Cell Membrane / Q000648:ultrastructure; D003208:Concanavalin A / Q000378:metabolism; D005293:Ferritins; D005613:Freeze Etching; D006023:Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011952:Receptors, Concanavalin A / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*
D006023:Glycoproteins; D008565:Membrane Proteins; D011952:Receptors, Concanavalin A; D003208:Concanavalin A; D005293:Ferritins