The organization of endoplasmic reticulum export complexes

NRK cells were infected with ts045 VSV as described above. After digitonin permeabilization (Plutner et al., 1992) and incubation in vitro as described in the Results, cells were fixed with 0.025% GA/3% paraforrnaldehyde for 30 rain. Cells were washed three times with PBS, quenched with 0.05 M glycine in 10% FBS/PBS for 30 rain, and incubated overnight with an anti-VSV-G cytoplasmic tail mAb (P5D4) (Kreis, 1986) . Cells were washed twice with FBS/PBS, followed by incubation with rabbit anti-mouse antibodies for l-2 h and with 6 or 10 nm gold conjugated to anti-rabbit antibodies for 2 h. Excess unbound antibodies were removed by washing, and the cells were fixed with 2.5% GA for 30 rain. Cells were scraped, pelleted, and processed for Epon embedding. For three-dimensional visualization using quick-freeze, deep etch replicas, immunolabeling was performed on glass coverslips.