Figure 3.

Requirements for ISG fusion. (a) Nucleotides (1 mM ATP and ATP-regenerating system, or 100 mM AMP-PNP) or ATP-depletion mixture (25 U hexokinase in 3 mM glucose) were added to the fusion reaction containing 100 μl of PNS from PC12 cells labeled for 30 min with [³⁵S]sulfate with or without PC2-ISGs. The reactions were incubated at 37° or 4°C for 30 min and then incubated for processing for 90 min. (b) Fusion requires trypsin sensitive proteins and p18 is generated in the lumen of membrane enclosed organelles. PC2-ISGs were pretreated with trypsin (0.01 and 0.1 mg/ml trypsin [final concentration] which corresponds to 20 and 200 ng trypsin/μg ISG protein) for 15 min at 37°C in the absence (lanes 3 and 4) or presence of STI (lanes 5 and 6). STI was added to the reactions shown in lanes 3 and 4, and fusion was assayed in a standard reaction by incubation for 30 min at 37°C, followed by processing for 45 min at 37°C. Alternatively, the reaction mixture was treated with the highest concentration of trypsin (0.1 mg/ml) for 15 min at 37°C after the processing reaction in the absence (lane 7) or presence (lane 8) of 0.3% Triton X-100. p18 was immunoprecipitated and then subjected to SDS-PAGE and autoradiography. Quantitation in a was performed by densitometry. The data shown are from a representative experiment done in duplicates.