Analysis of K. lactis cell-surface labeling by flow cytometry. (A) The K. lactis mutant mnn2-2 lacks terminal N-acetylglucosamine in its cell surface glycoproteins. Wild-type (MG1/2) and mnn2-2 (KL3) cells can therefore be differentially labeled by incubation with FITC-conjugated wheat-germ agglutinin (WGA), which has an affinity for N-acetylglucosaminyl residues. Phenotypic correction of the cell-surface defect was observed only in mnn2-2 transformants expressing wild-type SLC35A3. (Panel 1) Wild-type MG1/2 transformed with empty pE4 vector; (Panel 2) mutant mnn2-2 transformed with empty pE4 vector; (Panel 3) mutant mnn2-2 expressing the wild-type SLC35A3 gene; (Panel 4) mutant mnn2-2 expressing the mutated SLC35A3 gene with the V180F substitution. (B) Northern blot analysis. Total RNA extracted from the transformed yeast cells was separated by gel electrophoresis, blotted onto nylon membranes, and hybridized with a radioactively labeled full-length SLC35A3 cDNA probe. Positions of the 26S and 18S ribosomal RNA are indicated. (Lane 1) Wild-type MG1/2 transformed with empty pE4 vector; (lane 2) mutant mnn2-2 transformed with empty pE4 vector; (lane 3) mutant mnn2-2 expressing the wild-type SLC35A3 gene; (lane 4) mutant mnn2-2 expressing the mutated SLC35A3 gene with the V180F substitution. Ethidium bromide stained 26S and 18S rRNA to control for equal loading is shown below the Northern blot.
