Figure 5.
The nature of the signal sequence and its distance from Cub determine the extent of cleavage of Cub-R in the presence of Nub-Sec62p. (A) S. cerevisiae expressing Mfα37-Cub-Dha (construct 8; Figure 2) (lanes a and b), Mfα65-Cub-Dha (construct 9) (lanes c and d), Suc223-Cub-Dha (construct 10) (lanes e and f), Suc233-Cub-Dha (construct 11) (lanes g and h), Suc259-Cub-Dha (construct 12) (lanes i and j) and Suc2518-Cub-Dha (construct 13) (lanes k and l) were labeled with 35S-methionine for 5 min. The extracted proteins were either mock-treated (lanes a, c, e, g, i, and k) or treated with EndoH (lanes b, d, f, h, j, and l), followed by immunoprecipitation with anti-ha antibody and SDS-PAGE. (B) Same as panel A but the cells also contained Nub-Sec62p in addition to the Cub-fusions Mfα37-Cub-Dha, Mfα65-Cub-Dha, Suc223-Cub-Dha, Suc233-Cub-Dha, Suc259-Cub-Dha, Suc2518-Cub-Dha (lanes a–f). The analysis was carried out by immunoblotting whole- cell extracts with the anti-ha antibody. (C) S. cerevisiae cells expressing Suc223-Cub-Dha (construct 10; Figure 2) (lanes a–c) and Suc259-Cub-Dha (construct 12; Figure 2) (lanes d–f) together with either Nub-Sec62p (lanes b and e), Nub-Bos1p (lanes c and f) or the vector (lanes a and d) were labeled for 5 min with 35S-methionine. Whole-cell extracts were immunoprecipitated with anti-ha antibody, followed by SDS-PAGE and autoradiography. (D) S. cerevisiae cells expressing ΔSuc223-Cub-Dha (construct 15; Figure 2) or ΔSuc259-Cub-Dha (construct 16; Figure 2) together with either the vector (first six lanes) or Nub-Sec62p (last six lanes) were labeled for 5 min with 35S-methionine and chased for 10 and 30 min, followed by extraction, immunoprecipitation with anti-ha antibody, and SDS-PAGE. Numbers 15 and 16 indicate the positions of the corresponding (uncleaved) Cub fusions.