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

SV24 cells were grown in 35-mm dishes to form subconfluant layers. The cells were depleted of meth_ionine in methionine-free MEM (MEM-met) containing 2.2 g/liter sodium bicarbonate, 10% FCS (dialyzed against PBS), 2 mM glutamine, 100 U/mi penicillin, 100/~g/ml streptomycin, and 10 mM Hepes. The ceils were pulsed for 10 rain with 300 #1 of the same medium containing 500 #Ci/ml 35S-labeled L-methionine (Amersham), and after the radioactive pulse, 2 mi of normal growth medium supplemented with 1.5 mg/ml nonradioactive L-methionine was added to prevent any further incorporation of [35S]methionine into newly synthesized proteins. After various times the chase was stopped by placing the dish on an ice cooled metal slab and washing the cells with ice-cold PBS after removing the medium. There was no increase in dimer concentration when a 10-rain pulsed (not chased) dish was allowed to stand for 1 h on an ice cooled metal slab before processing (data not shown). This control showed that cooling the samples to 0~ stopped any PDI catalyzed or spontaneous disulphide bond formation. All of the pulse-chase incubations described above were done at 37~ in 5% CO~.