PMEL Mutations Make Functional Amyloid Pathogenic Figure 6. PMEL TMD mutants generate fibrils with aberrant morphology. Immortalized melan-Ink4a melanocytes that stably express wild- type (a, d), TMinsWAP (b, e) or TMR625C (c, f) variants of hPMEL were fixed and processed for cryoimmunoelectron microscopy. Ultrathin cryosections were immunogold labeled for hPMEL using the hPMEL-specific NKI-beteb antibody and 10 nm protein A gold. Note that dense immunolabeling is observed on short immature fibrils (arrowheads) within stage I melanosomes from cells expressing all three hPMEL variants (upper panels, a–c), but that mature, elongated fibrils are immunogold labeled only in compartments (asterisks) within cells expressing the TMD mutants (lower panels, d–f). Also note the densely packed profile of the immunogold-labeled mature fibrils in cells expressing TMD mutant hPMEL as compared to the thick pigmented fibrils that lack labeling, which are found within stage III melanosomes in cells expressing wild-type hPMEL and which show some spacing between neighboring fibrils (bracket). Bars, 200 nm. doi:10.1371/journal.pgen.1002286.g006 periods of time and with modest expression levels, the fibrils formed by the TMinsWAP—and to a lesser degree the TMR625C variant hPMEL—impair pigmentation. Together, these results suggest that the mutations found in the dominant DW chicken PMEL and Silver horse influence the assembly of the fibrils into sheets, creating a tightly packed structure that may be inaccessible to pigment and/or that inhibits melanin biosynthesis. The Smoky chicken mutation prevents formation of aberrant PMEL fibrils The Smoky chicken is a recessive revertant of the DW allele, reflecting a second site mutation that results in deletion of four residues from the PKD domain (PKDDLVVT) (Figure 8A) [30]. In homozygous form, the Smoky allele imparts modest pigment dilution in the feathers—similar to the modest pigment dilution observed in the silver mouse [26], the Pmel -/- mouse [29] and the fading vision zebrafish [28]—as compared to the dramatic loss of pigment imparted by the dominant DW allele. To investigate how the PKDDLVVT mutation might reverse the DW phenotype, we created hPMEL variants with either the PKDDLVVT deletion alone or together with the WAP insertion (PKDDLVVT-TMinsWAP) as found in the Smoky chicken PMEL allele. The variants were expressed in HeLa cells, and their maturation, proteolytic processing, and trafficking were assessed by metabolic labeling/ pulse chase and immunoprecipitation analysis and by IFM. As shown in Figure 8B, introduction of the PKDDLVVT deletion, either by itself or in combination with the TMinsWAP insertion, impaired PMEL exit from the endoplasmic reticulum (ER) as shown by a decreased maturation into the fully glycosylated, post- Golgi P2 form. Furthermore, the PKDDLVVT and PKDDLVVT- TMinsWAP hPMEL forms were not efficiently processed by proprotein convertase cleavage into Ma and Mb fragments. These data are reminiscent of the effects of deletion of the entire PKD domain [16,22] and suggest that the PKDDLVVT deletion impairs PMEL maturation. Although the pulse chase data suggested that the PKDDLVVT mutation impairs PMEL exit from the ER, a fraction of the Golgi- modified P2 form did accumulate over time. To determine whether this fraction of ‘‘mature’’ PKDDLVVT PMEL is properly trafficked to late endosomal compartments, we analyzed its localization in HeLa cells by IFM using an antibody (HMB45) that only recognizes Golgi-modified PMEL [12,20]. Unlike wild- type hPMEL, this ‘‘mature’’ form of PKDDLVVT or PKDDLVVT- TMinsWAP hPMEL did not localize significantly to late endosomal compartments marked by LAMP1 (Figure 8C, central panels). PLoS Genetics | www.plosgenetics.org 8 September 2011 | Volume 7 | Issue 9 | e1002286