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Fig. 8. gyp5gyl1 cells display a distal secretion defect at 13°C. (A) Invertase expression was induced in WT and gyp5gyl1 cells cultured at 13°C, and invertase secretion was monitored as described in Materials and Methods. The % of secreted invertase is the ratio (external invertase/total invertase). Values are the average of three experiments performed on two independent clones. One invertase unit is defined as the enzyme quantity producing 1 µmol of glucose/minute/10⁷ cells. (B) Proximal steps of secretory pathway are normal in gyp5gyl1 cells. Pulse-chase labelling and immunoprecipitation of CPY was performed on WT and gyp5gyl1 cells cultured at 13°C, as described in Materials and Methods. The ER (p1), Golgi (p2) and mature (m) forms of CPY are indicated. (C) Bgl2p-HA secretion assay was monitored as described in Materials and Methods. Equal amounts of cells were loaded on SDS-PAGE. After separation, proteins in the top of the gel were stained by Coomassie blue and proteins of the bottom of the gel were transferred onto nitrocellulose membrane. Immunodetection of this membrane with HA antibody was then performed. The control is one protein of the top of the gel stained by Coomassie blue.

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