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Fig. 2. Inhibition of glucose trimming slows ER exit rate of wild-type Shaker protein. (A,B) Wild-type Shaker protein was expressed in HEK293T cells, metabolically labeled for 10 minutes and chased for various times in the absence (-dNJ) or presence of 1 mM dNJ added during the starvation, pulse and chase periods (+dNJ) or during the chase period only (+dNJ^*). After detergent solubilization, the Shaker protein was immunoprecipitated and subjected to electrophoresis and fluorography. Representative fluorographs are shown; n=4. Open and closed arrowheads indicate the positions of the immature and mature forms of the Shaker protein, respectively. Open circles indicate the positions of two nonspecific background bands of variable intensity, which are also seen in untransfected cells upon immunoprecipitation with the Shaker antiserum (data not shown). A sharp, unstable band migrating near the mature form of the Shaker protein is also seen in untransfected cells (unmarked). Positions of molecular weight markers are indicated by bars (kD; kilodaltons). (C) The percentage of Shaker protein in the mature form has been plotted as a function of chase time. Data were obtained in the absence of dNJ (; t_1/2 for maturation=59 minutes), in the presence of dNJ added during the starvation, pulse and the chase (; t_1/2=101 minutes), or in the presence of dNJ added during the chase only (; t_1/2=57 minutes) (n=4). Here and in all subsequent figures, the data are presented as mean±s.e.m.

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