Figure 4.

Radiolabeled peptide sequence analysis to establish the NH₂-terminal processing status of intracellular species of a-factor. The a-factor species in immunoprecipitates from cells labeled with [³H]lysine (A), [³H]proline (B), or Trans³⁵S label (methionine:cysteine = 20:1) (C) were separated by 16% SDS-PAGE and transferred onto polyvinyldifluoride membrane. The intracellular a-factor intermediates P1, P2, and M were visualized by autoradiography, excised, and subjected to Edman degradation analysis. The radioactive material from each cycle was quantitated by scintillation counting as shown in the bar graph. Within the a-factor sequence, the radiolabeled amino acid used for labeling is shaded darkly, and the deduced P1→ P2 and P2→ M cleavage sites are indicated. The strain labeled in A and B is SM1762, which carries the wild-type a-factor plasmid pSM463 (2μ MFA1). The strain labeled in C is SM1932, which harbors an a-factor substitution mutant plasmid, pSM490 (2μ mfa1-I23M).