Figure 4.

Effect of GM130/giantin on cycling kinetics of p115. (A) Schematic of p115 constructs used to characterize membrane association. p115/1-934 lacks the acidic domain that contains the GM130/giantin-binding domain. p115/1-820 lacks the acidic domain and CC4. p115/1-766 lacks the acidic domain and CC3 and CC4. p115/1-655 lacks the acidic domain and CC1-CC4 that contains the SNARE-binding motif. AD, acidic domain. (B) HeLa cells were transfected with myc-tagged constructs diagrammed in A. After 24 h, cells were processed for double-label immunofluorescence with anti-myc and anti-ERGIC53 antibodies. All p115 constructs containing the SNARE-binding CC1 associate with membranes. p115/1-655 that lacks CC1 shows diffuse staining without discernible membrane association. Bar, 10 μm. (C) HeLa cells were transfected with p115/1-934-GFP. After 24 h, an area of the Golgi (arrows) was photobleached once, and fluorescence recovery into the bleached region was monitored by obtaining images every 3 s. Data from analogous experiments were analyzed and are presented in a graph in E. Bar, 10 μm. (D) HeLa cells were transfected with p115-YFP or p115/1-766-GFP. After 24 h, cells were processed for immunofluorescence with anti-GOS28 antibodies. Full-length p115 and p115/1-766 containing the SNARE-binding CC1 colocalize with GOS28. Bar, 10 μm. (E) Graph shows changes in fluorescence intensity over time. Data are presented as an average from at least 11 individual cells in at least two different experiments. Full-length p115 and p115/1-934 missing the GM130/giantin binding domain show analogous recovery.