Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

The immunofluorescence data shown in Fig. 3 suggest that a fraction of HBsAg may exist in a compartment which excludes ER markers, however, it does not allow one to distinguish between a gradual decrease in the concentration of ER marker relative to HBsAg and an abrupt change in the ratio of these proteins which would indicate a separate compartment. We quantitated the relationship between the distribution of HBsAg and ER markers by analyzing threedimensional double fluorescence data collected with confocal microscopy. Fig. 4 a shows a plot of the pixels in a single section of the confocal data resolved into their fluorescein (1D3-PDI-horizontal; shown separately in 4 c) and rhodamine (anti-HBsAg-vertical; shown separately in 4 b) intensifies. The strong, heavily populated diagonal corresponds to the bulk of the pixels in the image for which the ratio of PDI to HBsAg is relatively constant. The pixels which form a separate population at ~15 degrees from the vertical correspond to those regions of the image which are rich in HBsAg but have no detectable PDI. These regions are shown in Fig. 4 d in which only the pixels marked in Fig. 4 a were used to compose the image. The fact that this subpopulation of pixels is well separated in Fig. 4 a from the bulk of the pixels indicates that they correspond to a separate compartment Figure 2 . Distribution of ER marker proteins in SV24 cells. Indirect double immunofluorescence shows that the antibodies characterized in Fig. 1 produce typical ER staining patterns in paraformaldehyde-fixed SV24 cells. The antibodies used are anti-PDI (a, visualized with rhodamine) which shows an identical reticular staining pattern to 10C3 (b, visualized with fluorescein) and anti-KXsKDEL (c, visualized with rhodamine) which coincides with the staining for 1D3 (d, visualized with fluorescein). Structures that are morphologically similar to the HBsAg budding compartment and exclude ER markers are indicated by arrowheads. Bars, 10/~m. rather than reflecting the result of continuous variation in the intensity of either PDI or HBsAg staining. Processing of many sections of double stained cells revealed the presence of only the two populations of pixels seen in Fig. 4 a for transfected cells.