FIG. 3.

N-terminal requirements for prosequence cleavage. (A) Krp1 mRNA and mRNAs from various Krp1 mutants that lack the normal basic motifs at either the primary or internal site were translated in Xenopus egg extract for 1 h in the presence of [³H]leucine before being analyzed by SDS-PAGE on a low-percentage gel (lanes 1 to 9). Krp1[IA82][KA102] mRNA was also translated for 6 h before analysis in order to investigate the slow processing of this mutant (lane 10). The positions of molecular weight markers (in thousands) are shown on the left. (B) Selected samples were also analyzed on a high-percentage acrylamide gel (38). To aid in the interpretation of prosequence fragments, we prepared versions of Krp1 with stop codons introduced immediately after Arg102 (Krp1[R102*]), Arg99 (Krp1[R99*]), and Arg82 (Krp1[R82*]). Translation of these truncated proteins in extract resulted in translocation into microsomes and presequence cleavage to generate markers that are appropriate for different prosequences.