Figure 1.

Cav^DGV is targeted to the membrane of intracellular vesicular structures. a, BHK cells were transfected with HA-tagged cav-3^DGV for 24 h and were then labeled with antibodies to HA tag, followed by specific Cy3-conjugated secondary antibodies. Cav^DGV mainly located in enlarged intracellular rings, and some protein was detected in the tubular elements of the ER (see Fig. 2) and in the perinuclear region (arrows and g and h). b, BHK cells were transfected with HA-tagged cav^DGV for 24 h and were then treated with 5 μg/ml of cycloheximide for an additional 24 h. After blocking of protein synthesis, the truncated protein was exclusively detected in very enlarged intracellular vesicles (arrows). c, BHK cells were transfected with GFP-tagged cav^DGV and frozen sections were processed for immunoelectron microscopy using anti-GFP antibodies, followed by protein A–gold. The mutant protein was detected enriched on the membrane of electron-lucent vesicles of 300–500-nm diam, often surrounded by intermediate filaments (arrows). d and e, Lowicryl sections (of freeze-substituted/low temperature embedded sections) of GFP-tagged cav^DGV-transfected cells labeled with antibodies to GFP followed by protein A–gold. Cav^DGV-containing vesicles (CDV) comprise a membrane bilayer (see arrows in d and e) surrounding a putative lipid-filled area. f, Epon sections showing a morphologically similar structure connected to tubular elements possibly corresponding to the ER (arrow and arrowhead), which were frequently observed in nontransfected cells. The typical morphology of lipid-filled body enclosed by a bilayer structure (see insert) is readily observed in epon sections. g, BHK cells were cotransfected with myc-tagged sialotransferase (a specific marker for trans Golgi) and HA-tagged cav^DGV and the distribution of the proteins studied by immunofluorescence by means of antitag specific antibodies. The distribution of sialotransferase (green) and cav^DGV (red) clearly overlapped in the Golgi area, however CDV rings did not contain the Golgi marker. h, HA-tagged cav^DGV-transfected cells were label with an mAb to cav-1 (which exclusively recognizes the Golgi complex conformation of cav-1) and a polyclonal antibody to HA-tag. When cav^DGV (red) accumulated in the Golgi region, the truncated protein clearly colocalized with the Golgi complex-associated pool of endogenous cav-1 (green). i and l, HA-tagged cav^DGV-transfected cells were labeled with polyclonal antibodies to the peroxisome marker SKL (l) or the late endosome marker, lysobisphosphatidic acid (h) and with an mAb to the HA-tag. The endosome and peroxisome markers (green) were excluded from the CDV compartment (red). j, Cav^DGV-transfected cells were incubated for 2 h at 37°C with 10 μg/ml of GM1 to allow insertion of the ganglioside into the plasma membrane and internalization. GM1, detected by using 1 μg/ml of Cholera toxin–FITC, was completely excluded from the CDV compartment (red). k, BHK cells were cotransfected with Niemann-Pick C1 protein and cav^DGV for 24 h and were then labeled with a polyclonal antibody to NPC1 and the mAb to the HA tag. Transfected NPC1 (green) does not colocalize with the CDV vesicles (red). Bars: (a, b, and g–l) 5 μm; (c–f) 100 nm.