Figure 4.

Golgi-localized DAB precipitation inactivates VSV-G secretion. (A) HeLa-ManII-HRP cells were transfected with GFP-VSV-G^ts045 encoding plasmids and incubated overnight at 40°C. Cells were then shifted to 32°C without (a–d) or with (e–h) pre-treatment with DAB/H₂O₂ and fixed in paraformaldehyde. Cells were processed for immunofluorescence (see Materials and Methods) to stain plasma membrane–localized VSV-G proteins (in red) and the Golgi complexes (in blue), whereas the bulk of GFP-VSV-G protein is directly visible in green. As expected, in the absence of Golgi cross-linking, GFP-VSV-G^ts045 is found in the ER just before shifting at permissive temperature (a) and then reaches the Golgi apparatus (b) before being expressed at the plasma membrane (c and d). In contrast, after cross-linking the Golgi with DAB/H₂O₂, GFP-VSV-G^ts045 exits the ER at permissive temperature and reaches the Golgi region (e and f) but is barely visible at the plasma membrane even after 120 min (h). Bar, 20 μm. (B) An experiment similar to the one described in A was analyzed by FACS to quantify the arrival of VSV-G^ts045 at the plasma membrane. As observed in A, whereas in nontreated cells VSV-G is efficiently transported at the plasma membrane, cross-linking of the Golgi strongly reduces its transport to the plasma membrane. (C) A similar experiment was carried out but using pulse chase and immunoprecipitation of VSVG-GFP proteins followed by de-glycosylation, SDS-PAGE separation, and PhosphorImager-based quantification. VSV-G is blocked in a pre-Golgi compartment in cross-linked cells, as indicated by the persistence of EndoH sensitivity of its glycosylation.