Figure 8.

The Golgi complex in cells lacking GCC185 is functional. (A) Arrival of VSV-G-YFP protein at the surface of control (mock transfected) or GCC185 siRNA–treated cells. Cells were pulse labeled for 30 min and then chased for the times indicated; surface VSV-G-YFP was determined using a surface antibody binding procedure. Data shown are from two separate experiments in which cells were chased for 45 or 120 min, respectively. No surface VSV-G protein was detected in reactions held on ice. Values for surface VSV-G from PhosphorImager scans were normalized for protein content and are presented as percentage of control reactions; background at time 0 chase was subtracted. Examples of the GCC185 depletion blots are presented in the inset at the top. (B) Tyrosine sulfation of endogenous acceptors was determined in equal quantities of control (mock-transfected) and GCC185-depleted cells. PhosphorImager scans yielded 3280 arbitrary units for the GCC185 control and 4360 arbitrary units for the Golgin-97 control reaction. Data are normalized to 100% to ease comparison. (C) Cellular content of fluorescent transferrin internalized for 5 and 15 min determined by ImageJ software, with (dashed line) or without (solid line) GCC185 depletion.