Fig. 6. Expression of EGFP-Myo1b delays the processing of Pme17. (A) Schematic representation of Pmel17 and the processed forms proposed by Berson et al. (Berson et al., 2001). Pmel17 is synthesized as type-I integral membrane protein precursor P1 in the endoplasmic reticulum, glycosylated to precursor P2 in the Golgi, and cleaved into two disulfide-linked subunits: a large lumenal subunit M and an integral membrane subunit Mß. Antibodies HMB45 and HMB50 recognize the N-terminus of P1, P2 and M. HMB50 immunoprecipitates both M and Mß, because of their disulfide-link. PEP13 antibody recognizes the C-terminus of P1, P2 and Mß. (B) Lysates from five million EGFP-expressing or EGFP-Myo1b-expressing cells were immunoprecipitated with HMB50. Precipitates were then analysed by western blotting with HMB45 to detect M, and PEP13 to detect P1, P2 and Mß. The same membrane was used for the detection of the different polypeptides. One of four representative experiments is shown. (C) EGFP-expressing cells and EGFP-Myo1b cells were metabolically pulse-labelled with [³⁵S]methionine/cysteine for 10 minutes and then chased for 0 minutes, 30 minutes, 1 hour and 2 hours. 25 µCi of cell lysate were then immunoprecipitated with HMB50. Precipitated proteins were analysed by SDS-PAGE and detected by autoradiography. (D) The relative intensities of Mß in EGFP and EGFP-Myo1b cells were quantified and expressed in arbitrary units. (C,D) One of three representative experiments is shown.