Green fluorescent protein-cell wall fusion proteins are covalently incorporated into the cell wall of Saccharomyces cerevisiae.
Tagging of two cell wall mannoproteins, Cwp1p and Cwp2p, in Saccharomyces cerevisiae with the green fluorescent protein from Aequorea victoria resulted in incorporation of fluorescent fusion proteins into the cell wall. Both living cells and isolated cell walls became brightly labeled. Intriguingly, the incorporation patterns of both fusion proteins differed. Western analysis of enzymatically released fusion proteins showed that they were covalently linked to the beta-1,6-glucan part of the cell wall. Removal of the glycosylphosphatidyl-inositol anchor signal sequence of the green fluorescent protein-cell wall protein fusion proteins resulted in secretion of the proteins into the culture medium. These results indicate that green fluorescent protein-cell wall protein fusion proteins can be used as a convenient fluorescent marker to study the incorporation of specific cell wall proteins and the control mechanisms involved.
Ram
A F
AF
Institute for Molecular Cell Biology, Fungal Cell Wall Group, University of Amsterdam, BioCentrum Amsterdam, The Netherlands.
Van den Ende
H
H
Klis
F M
FM
D015153:Blotting, Western; D002473:Cell Wall / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C093810:CWP1 protein, S cerevisiae; C093811:CWP2 protein, S cerevisiae; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins