Figure 2.

GRASP55 depletion severs the connections between Golgi stacks and inhibits normal protein glycosylation. (A) Representative cells stably expressing GalNAcT2-GFP were tested for Golgi integrity by photobleaching a Golgi segment with a brief laser pulse and measuring the return of Golgi fluorescence to the bleached spot. Bar, 10 μm. Corresponding movies are provided (see Supplemental Figures S1 and S2). (B) Fluorescence recovery was quantified by dividing GFP fluorescence within the bleached spot by the fluorescence within a nearby unbleached region in the same Golgi object, after subtracting background fluorescence from both. Each curve was further normalized by bracketing values between a minimum of 0 and a maximum of 1 (n = 3, ≥12 cells each, ±SEM). (C) To test the glycolytic processing of Golgi cargo, live, nonpermeabilized, control- or GRASP55-depleted cells were stained with fluorescently tagged GSII lectin and then rinsed, fixed and processed for immunofluorescence by using GP73 as a Golgi marker. Bar, 4 μm.