Author Summary Amyloid is a protein fold that is normally associated with pathology, such as neurodegeneration in Alzheimer, Parkinson, and Creutzfeldt–Jakob diseases. The amyloid fold has also been exploited by nature for functional purposes; for example, proteolytic fragments of the pigment cell-specific integral membrane protein, PMEL, form amyloid fibrils upon which melanin pigments polymerize within subcellular organelles called melano- somes. Whereas animal models that entirely lack PMEL expression have modest pigment loss, chickens or horses with small in-frame mutations that alter a non-amyloido- genic region of PMEL have severe pigment loss that is thought to be associated with pigment cell death. We show here that these mutations alter the capacity of this region to self-associate, likely changing the conformation of full-length PMEL oligomers. While these changes do not affect the intrinsic ability of PMEL to form amyloid fibrils, they alter either the fibrils themselves or the kinetics of fibril formation such that they form more compact structures and inhibit melanin formation when expressed in melanocytes in culture. Based on our results, we speculate that minor alterations in pre-amyloid assembly of an amyloidogenic protein influences entry into either a benign or a pathogenic amyloid pathway. intermediates to facilitate their detoxification, polymerization, and/ or intercellular or intracellular transfer [8]. This function seems to be important for optimal pigmentation, as animal models with apparently hypomorphic mutations in PMEL show varying levels of hypopigmentation, ranging from modest in the silver mouse [26] to more pronounced in Merle dogs [27] and fading vision zebrafish [28]. In the accompanying paper by Hellstro¨m et al. [29], we show that a complete loss of PMEL expression in the Pmel -/- mouse also presents with a modest pigment dilution. Compared to the more modest effects of the PMEL mutations described above, genetic models in the chicken and horse show that in some cases, PMEL mutations can result in severe hypopigmentation. In the chicken, a nine bp in-frame insertion PMEL Mutations Make Functional Amyloid Pathogenic within the coding region for the PMEL transmembrane domain (TMD; TMinsWAP) is associated with the nearly complete loss of feather eumelanin in Dominant White (DW) chickens [30]. The pigment loss in DW chickens is associated with poor melanocyte survival in culture and with melanocyte depletion along the feather shaft in vivo [31], both exaggerations of the increased cell cycle length [32] and slow, progressive melanocyte loss [33] observed in homozygous mice bearing the recessive, hypomorphic PMEL silver mutation. However, unlike mouse silver, the DW allele is associated with impaired melanosome maturation and with melanosome loss within epidermal melanocytes [34,35]. Moreover, the DW allele differs from mouse silver and other PMEL hypomorphs in that it confers hypopigmentation in a dominant fashion, suggesting that the DW PMEL product must either inhibit endogenous PMEL function in a dominant-negative manner or confer some gain-of- function that is detrimental to melanocyte function and/or health. Interestingly, a concomitant deletion within the conserved lumenal Polycystic Kidney Disease-1 homology (PKD) domain in the Smoky chicken (PKDDLVVT) reverts the dominant phenotype of the TMinsWAP DW mutation, resulting in a much more modest hypopigmentation that is inherited in a recessive manner [30]. In Silver horses (HoSi), a dominant missense mutation in PMEL results in a transversion that substitutes a cysteine for the second of three consecutive arginine residues immediately following the PMEL TMD (TMR625C), causing a dilution of black pigment that is most noticeable in the mane and tail of the animal [36,37]. An orthologously identical TMR625C mutation in the Dun chicken PMEL allele is accompanied by an additional gene deletion (TMD5) that eliminates five residues from the PMEL TMD [30]. However, while Dun is likewise dominant, it confers a more modest hypopigmentation, suggesting either species-specific variation in the TMR625C PMEL phenotype or that the TMD5 secondary mutation might partially dampen the pigmentation defect that the TMR625C mutation confers individually. Why the mutations in the DW chicken and Silver horse present a dominant phenotype and how they might be reverted by the additional deletions in the Smoky and (perhaps) Dun chickens is not understood. Given the severity of their pigmentation phenotype, the association with loss of melanosome integrity and melanocyte Figure 1. Schematic diagram of PMEL proteolytic processing. Diagram of PMEL domain structure, relevant proteolytic fragments and proteolytic cleavage sites (dashed lines and scissors). Within the primary sequence are indicated the signal peptide (S), N-terminal region (NTR), polycystic kidney disease repeat domain (PKD), repeat domain (RPT), transmembrane domain (TM) and cytoplasmic domain (Cyto). The signal peptide is removed from the N-terminus of PMEL in the ER by signal peptidase (SP). PMEL is cleaved by a proprotein convertase (PC) to produce Ma and Mb fragments, which are linked by disulfide bonds (not shown). Subsequent cleavage by a site 2 protease (S2P) at a site proximal to the lumenal side of the TM produces a C-terminal fragment (CTF) and liberates Ma. As yet unidentified enzymes (marked ‘‘??’’) further cleave Ma at as yet unknown sites to produce MaC. MaC fragments and a fragment of the PKD domain have been shown to accumulate in fibrillar stage II melanosomes [23]. doi:10.1371/journal.pgen.1002286.g001 PLoS Genetics | www.plosgenetics.org 2 September 2011 | Volume 7 | Issue 9 | e1002286