FIGURE 8:

The NTR and the PKD must be provided in cis for fibril formation but can stabilize the RPT domain–containing MαC fragments in trans. (A) Schematic representation of the PMEL mutant ΔRPT. (B, C) Mel220 cells expressing wt-PMEL or ΔRPT were analyzed by IF using antibodies against the early endosomal marker EEA1 (ab70521) and mature PMEL (HMB50; B) or the lysosomal marker LAMP1 (H4A3) and mature PMEL (HMB50; C). (D) SDS-lysed total membranes derived from cells expressing wt-PMEL, ΔRPT, or D73K or coexpressing ΔRPT and D73K were analyzed by Western blot using the PMEL-specific antibodies Pep13h, HMB45, and I51. (E) Mel220 transfectants stably expressing ΔRPT were analyzed by EM (Epon-embedded cells; middle) or cryo–immuno EM using the PMEL-specific antibody HMB50 (top and bottom). (F) EM analysis of Epon-embedded Mel220 transfectants expressing ΔRPT. Quantification of fibril formation (n = 15) is shown. (G) D73K-derived MαC and mature RPT domain stabilized by a coexpressed ΔRPT construct are distributed in the Triton X-100–insoluble fibril fraction, whereas D73K-derived Mα remains Triton X-100 soluble. A total membrane fraction was extracted in 1% Triton X-100 for 1 h and centrifuged at 100,000 × g for 45 min before supernatant was removed and analyzed by SDS–PAGE and Western blotting (left lane labeled Tx100). The Triton X-100–insoluble pellet was resuspended in PBS/1% SDS/1% β-mercaptoethanol and incubated for 10 min at room temperature, followed by 10 min at 100°C and analyzed on the same gel (right lane labeled SDS). (H) Cells expressing wt-PMEL or D73K or coexpressing ΔRPT and D73 were analyzed by IF using the PMEL-specific antibodies Pep13h and HMB45. (I) The indicated synthetic peptides were adsorbed to nitrocellulose membrane, and a dot blot was performed using antibody I51. Note that the exchange of threonine 210 to methionine abrogates the recognition of the peptide by the antibody (compare lane 1 to lane 5). (J) SDS-lysed total membranes derived from cells expressing wt-PMEL or T210M were analyzed by Western blot using the PMEL-specific antibody I51. (K, L) Mel220 cells expressing T210M were analyzed by IF using antibodies against the early endosomal marker EEA1 (ab70521) and mature PMEL (HMB50;K) or the PMEL-specific antibodies Pep13h and HMB45 (L). (M) Mel220 transfectants stably expressing T210M were analyzed by EM (Epon-embedded cells). (N) SDS-lysed total membranes derived from cells expressing wt-PMEL or T210M were analyzed by Western blot using the PMEL-specific antibody HMB45. (O) SDS-lysed total membranes derived from cells expressing D73K alone, coexpressing ΔRPT and D73K, or coexpressing I51-nonreactive ΔRPT-T210M and D73K were analyzed by Western blot using the PMEL-specific antibodies ab52058, HMB45, and I51.