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Fig. 6. Golgi function is impaired in 
ypt1/SLY1-20 cells. (A) Glycosylation is enhanced in ypt1/SLY1-20 cells. Strains were grown overnight at 23°C to early to mid log phase and shifted for 1 hour to 37°C where indicated. Total yeast lysates were prepared and equal amounts of protein were separated by SDS-PAGE. Glycosylated proteins were visualized with a Pro-Q Emerald 300 Kit. More glycoproteins were detected in ypt1/SLY1-20 cells irrespective of the temperature. M, marker; pos. con., positive control (HRP); neg. con., negative control, Sec24p; GM, CandyCane glycoprotein molecular mass standards: (from top to bottom) 180 kDa; 82 kDa; 42 kDa; 18 kDa. (B) CPY outer chain glycosylation still occurs at 23°C and is abolished 37°C in ypt1/SLY1-20 cells. A pulse-chase experiment was performed at either 23°C or 37°C. Transport of CPY to the vacuole was delayed, yet the glycosylation occurred normally at 23°C in ypt1/SLY1-20 cells. At 37°C transport between the ER and the Golgi is blocked. Hence no CYP maturation could occur. Note that a wild-type strain expressing SLY1-20 behaved like the wild type in the assay.
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