FIGURE 5:

ARF1^EN-Halo post-Golgi tubules contain the anterograde cargo VSV G-SNAP but not the raft-associated cargo GPI-GFP-FM4. ARF1^EN-Halo cells were electroporated with GFP-FM4-GPI (a–c) and VSV G-SNAP (d, e) encoding plasmids. Cells were labeled with SiR-CA only (a–c) or 505-BG and SiR-CA (d, e). (a) Aggregated GFP-FM4-GPI was released from the ER by adding the disaggregating drug. (a–c) GFP-FM4-GPI was never observed in ARF1^EN-Halo-positive structures. (b, c) ARF1^EN-Halo tubules devoid of GFP-FM4-GPI are highlighted by arrows. (d) VSV G-SNAP cells were grown overnight at 40.5°C and then shifted to 32°C on the microscope stage to release the cargo from the ER. (e) At ∼30 min after shifting the cells to the permissive temperature, tubules containing both ARF1^EN-Halo and VSV G-SNAP were observed forming at the Golgi (arrows). (f) A negligible fraction of ARF1^EN-Halo tubules contained the cargo GFP-FM4-GPI. The frequency of VSV G–positive tubules exiting the Golgi is 3 ± 1 tubules/min, and 80 ± 13% of the ARF1^EN-Halo tubules also contained VSV G–SNAP (g). Error bars represent SD. Scale bars, 10 μm (a, c), 2 μm (b, d, e).