Glycosylation increases potassium channel stability and surface expressionin mammalian cells

N-linked glycosylation is not required for the cell surface expression of f unctional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L ., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607 -5613). We have now investigated whether glycosylation increases the stabil ity, cell surface expression, and proper folding of Shaker protein expresse d in mammalian cells. The turnover rates of wild-type protein and an unglyc osylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In co ntrast, the unglycosylated mutant was rapidly degraded (t(1/2) = similar to 18 h). Lactacystin slowed the degradation of the mutant protein, implicati ng cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive deg radation could be conferred on wild-type Shaker by a glycosylation inhibito r. Expression of the unglycosylated mutant on the cell surface, assessed us ing immunofluorescence microscopy and biotinylation, was dramatically reduc ed compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfide-bonded adducts was q uantitatively similar in the wildtype and unglycosylated mutant proteins. O ur results indicate that glycosylation increases the stability and cell sur face expression of Shaker protein but has little effect on acquisition of t he native structure.