Fig. 4. CPY transport and maturation were monitored by pulse-chase analysis. (A) Strain APY026 gea1-19 and wild-type strain CJY049-11-1 were incubated at 37°C for 30 minutes, pulse-labeled for 10 minutes, then chased for the times indicated. CPY was recovered from cell extracts by immunoprecipitation and visualized by SDS-PAGE and fluorography. (B) A pulse-chase regime identical to that described in part (A) was carried out for APY022 gea1-6 and the corresponding wild-type strain (CJY049-3-4), followed by two successive immunoprecipitations, first using anti-CPY, then anti-1,6-antisera. (C) Strain CJY062-10-3 gea1-4 and CJY049-3-4 (wildtype) were subjected to a pulse-chase regime as described in part (A) except that preincubation was carried out at 38°C for 30 minutes. Single CPY immunoprecipitations were carried out. (D) After a first immunoprecipitation with anti-CPY antibodies as described in part (A) for strains APY026 gea1-19 and CJY049-11-1 (GEA1), a subsequent immunoprecipitation was carried out using anti-1,6-antiserum. ‘mCPY’ refers to the mature vacuolar form; ‘p’ indicates a precursor form; p1 is the ER-core-glycosylated precursor; p2 is the fully glycosylated Golgi form; p and pß are ER precursor forms because they do not contain 1,6-linkages. ‘p1*’ is a precursor form found in the gea1 mutants that does not correspond to p1, p2 or mCPY as seen in a wild-type strain; it is a Golgi form since it is precipitated by anti-1,6-antiserum. (E) Percentages of precursor forms (all forms grouped) and of mature vacuolar CPY for wildtype and each gea1 mutant, as determined by quantitative Phosphorimager analysis. The values obtained for the amount of mCPY for the wild-type strain in three separate experiments were 64%, 67% and 69%: the average is shown.