FIG. 5.

Newly synthesized FHV protein A rapidly associates with cellular membranes in Drosophila S2 cells. (A) Cells were incubated with PBS and 0.01% saponin on ice for 10 min and centrifuged at 10,000 × g for 5 min to recover the supernatant fraction (S) and the resultant pellet, which was extracted in an equal volume of RIPA buffer and centrifuged at 10,000 × g for 5 min to remove nuclei and insoluble cellular debris and to recover a final pellet (P) fraction. Equal-volume fractions were separated by SDS-PAGE and immunoblotted with antibodies against the cytosolic proteins Hsp83 and tubulin, the mitochondrial membrane protein VDAC, the mitochondrial matrix protein Hsp60, and FHV protein A (PtnA). (B) Cells stably transfected with pS2FA or pS2LacZ were induced with Cu²⁺, incubated with 125 μCi per ml [³⁵S]Met-Cys, and harvested at 15, 30, 45, 60, and 90 min. Cells were separated into cytosolic and membrane fractions as described above, and FHV protein A and β-galactosidase were immunoprecipitated with HA-specific antibodies and analyzed by fluorography. (C) Cells stably transfected with pS2FA were induced and labeled as described above for 90 min in the presence or absence of 1 μM geldanamycin (GA) and separated into cytosolic (C) and membrane (M) fractions. Full-length FHV protein A was immunoprecipitated and analyzed by fluorography. Results are representative of at least four independent experiments.