PMEL Mutations Make Functional Amyloid Pathogenic that field; at least 60 fields of equal size/magnification were analyzed, containing more than 400 melanosomes of different maturation stages. *, p ,0.05; **, p ,0.01 as determined by ANOVA and Student’s t-test. D. Quantification of the number of pigmented granules (Stage III-IV) per mm2 of cell area in cells expressing wild-type, TMinsWAP or TMR625C hPMEL variants or cells transduced with virus that did not co-express hPMEL. Each measurement is shown individually; median values are indicated. **, p,0.01 as determined by ANOVA and Student’s t-test. doi:10.1371/journal.pgen.1002286.g007 Rather, it partially overlapped with endocytic recycling compart- ments labeled by internalized transferrin (Figure 8C, right panels), much like PMEL lacking the entire PKD domain [16]. This indicates that the PKDDLVVT deletion impairs not only PMEL maturation in the early biosynthetic pathway but also its selective incorporation into ILVs for trafficking to late endosomal compartments, thus precluding access to proprotein convertase cleavage [16]. Consistent with the requirement for sorting to ILVs and proprotein convertase cleavage for fibril formation, MaC fragments are not detected in detergent-insoluble fractions of cells expressing the PKDDLVVT or PKDDLVVT-TMinsWAP mutants (Figure S5). We conclude that the PKDDLVVT mutation reverts the dominant phenotype of the TMinsWAP mutation by impairing access of PMEL to fibrillogenic compartments and blocking aberrant amyloid fibril formation. The results described above suggest that primary PMEL mutations that inhibit pigmentation in a dominant fashion can be reverted by second site mutations that prevent the aberrant PMEL variants from accumulating in fibrillogenic compartments. The dominant but milder Dun chicken PMEL allele contains both the orthologous TMR625C mutation found in the HoSi allele and an additional deletion of 5 amino acids within the TMD (TMD5; Figure S6A). We therefore predicted that the TMD5 mutation, like the Smoky-associated PKDDLVVT mutation, would impair PMEL accumulation within fibrillogenic endosomal compartments. Consistent with this prediction, when expressed in HeLa cells, introduction of the TMD5 mutation in the context of hPMEL decreases the expression level of all mature (post-ER) PMEL species, including the fibrillar MaC forms (Figure S6B), even when corrected for mRNA expression (data not shown). This likely reflects enhanced ER-associated degradation due to poor folding or membrane incorporation, as the material that does exit the ER appears to be processed with normal kinetics (Figure S6C-S6D). These data suggest that while the TMD5 mutation does not prevent delivery of PMEL to fibrillogenic compartments (Figure S6E), it can impair the efficiency with which aberrant PMEL fibrils accumulate within such compartments. Given that the primary effect of the TMD5 mutation is on ER exit, it is highly likely to have similar effects in the context of the TMR625C mutation, as in Dun chickens, which does not affect ER exit (Figure 3). Discussion Unlike most commonly known forms of amyloid, which are thought to provoke pathologic processes, PMEL is an example of a benign and functional amyloid. Here we show how mutations in the PMEL TMD are associated with an aberrant amyloid fibril biogenetic pathway, altering the normally physiological amyloid to produce a pathological form that impairs pigmentation within melanocytes. Epidermal melanocytes from animals harboring these mutations are depleted of melanosomes [34] and have decreased viability in vitro and perhaps in vivo [31], suggesting that the formation of these aberrant fibrils impairs melanosome integrity and may be toxic to the pigment cell. Although the TMD does not form part of the amyloid core, mutations in this domain influence TMD oligomeric properties that reverberate distally on the association between the amyloidogenic domains of PMEL, as evidenced by an abnormal packing of the mutant PMEL fibrils. We also show that secondary mutations found in animals in which the pigment dilution associated with the primary pathogenic TMD mutations are dampened or reverted prevent the accumulation of these PMEL isoforms in fibrillogenic compart- ments, thus mimicking a PMEL knockout. This finding indicates that it is less detrimental to express no fibrils at all than to express aberrant fibrils that inhibit pigmentation and might be toxic to the melanocyte. Interactions among TMDs are known to influence multisubunit complex assembly and function in vivo [43–48]. Here, we show that whereas the TMD of hPMEL normally does not promote oligomerization, introduction of either the TMinsWAP or the TMR625C mutations found in the DW chicken or Silver horse PMEL orthologues results in substantial oligomerization potential. Oligomerization by the PMEL TMD was similarly enhanced by insertion of three leucine residues in place of the DW-associated WAP insertion, suggesting that the effect reflected increased TMD length rather than specific amino acid side chain interactions. Although the observed increase in dimerization by the TMR625C could not be directly compared to the wild-type TMD with the extended cytosolic domain because the latter did not insert properly into the E. coli plasma membrane, a similar degree of oligomerization was observed upon alteration of R625 to serine as with the HoSi-associated cysteine, but not to lysine, all of which supported proper membrane insertion. This suggests that increased oligomerization mediated by the TMR625C mutation reflects removal of a positive charge from the TMD boundary, decreasing the electrostatic repulsion between neighboring PMEL molecules by the membrane proximal arginine triplet. Interest- ingly, the TMR625C mutation was associated with greater CTF stability. In addition, whereas we often observed a CTF doublet for both wild-type and TMinsWAP PMEL, reflecting the two possible S2P sites [18], we always detected a single TMR625C CTF species. The altered TMD mediated oligomerization of this mutant might thus result in either a greater accessibility of one site over the other or aberrant partitioning of PMEL to membrane subdomains that preferentially harbor a site-specific enzyme, akin to what has been proposed to occur between a- and b-secretases in the cleavage of APP to produce pathologic Ab [49]. How increased TMD-mediated dimerization might influence PMEL folding, assembly, and fibril formation is not yet clear. A previous study found that the DW chicken PMEL associated with membrane microdomains to a similar degree as wild-type hPMEL [38], suggesting that the TMinsWAP mutation does not alter membrane partitioning. The same study suggested that matura- tion and proteolytic processing of DW chicken PMEL was not substantially different from that of wild-type hPMEL. Consistently, we find that neither the TMinsWAP nor the TMR625C mutation affect hPMEL biosynthetic trafficking, proteolytic maturation, delivery to ILVs within endosomes, or the initial stages of protofibril formation. While we could not detect a previously described effect of the TMinsWAP mutation (or of the TMR625C mutation) in reducing disulfide bond-mediated dimerization of the PMEL lumenal domain [38]—which likely reflected more a lack of heteromeric interactions between chicken and human PMEL than an effect of the TMD mutation itself—it is highly likely that the induced TMD interactions impact the orientation and PLoS Genetics | www.plosgenetics.org 10 September 2011 | Volume 7 | Issue 9 | e1002286