PMEL Mutations Make Functional Amyloid Pathogenic TMinsWAP mutation. B. Metabolic labeling/pulse chase analysis of HeLa cells transiently transfected with wild-type, PKDDLVVT, TMinsWAP or PKDDLVVT- TMinsWAP variants of hPMEL. Cells were labeled, chased and Triton X-100-soluble cell lysates were immunoprecipitated with an antibody to the hPMEL C-terminus as in Figure 3C. Note that variants bearing the PKDDLVVT deletion, regardless of the presence of the TMinsWAP insertion, do not mature efficiently to P2 and fail to accumulate Ma and Mb fragments. C. IFM analysis of HeLa cells transiently expressing wild-type (a–c), PKDDLVVT (d–f) or PKDDLVVT-TMinsWAP (g–i) variants of hPMEL. Cells were fixed after exposure for 30 min at 37uC to fluorophore-conjugated transferrin (Tf) to label early sorting and recycling endosomes, then stained with an antibody that recognizes only mature PMEL (HMB45) and an anti-LAMP1 antibody. Left panels (a, d, g) show PMEL labeling only; middle panels (b, e, h) show overlap of LAMP1 (green) and HMB45-labeled PMEL (pseudocolored red); and right panels (c, f, i) show overlap of internalized Tf (green) and PMEL (red). Insets show a 4X magnification of the boxed areas. Note that wild-type PMEL overlaps with LAMP1 but not internalized Tf, whereas PKDDLLVT overlaps partially with Tf but not LAMP1. doi:10.1371/journal.pgen.1002286.g008 proximity of PMEL dimers that form early in PMEL biosynthesis [13,38]. Although the induced conformational changes are likely subtle and do not impact recognition by the ER quality control system, biosynthetic trafficking, or the ability to form fibrils, they do appear to have downstream effects on the assembly of fibrils into sheets and/or in the packing of the sheets. One potential explanation for these effects is that non-amyloidogenic domains of PMEL dimers that protrude from the fibrils and regulate the packing of fibrils into higher order assemblies might be positioned differently. An alternative explanation is that oligomerization via the PMEL TMD might increase the kinetics of higher order fibril assembly. Either effect might result in more tightly packed fibrils within early stage melanosomes. How might the TMinsWAP or TMR625C PMEL variants impair melanogenesis and melanosome integrity? If indeed increased TMD oligomerization translates a conformational change to the lumenal domain to alter either the mode or kinetics of fibril polymerization into sheets, several mechanisms could be envisioned. Both altered conformation or kinetics—either by physical blockade through tighter packing or by overly rapid kinetics of sheet assembly—would potentially preclude the delivery of melanogenic enzymes, such as tyrosinase, to the lumen of the maturing melanosome [50]. This would in turn have the effect of concentrating the formation of oxidative melanin intermediates at the limiting membrane of the maturing melanosome and subjecting the limiting membrane to oxidative attack, potentially damaging the integrity of the organelle. This would explain both the loss of melanization (this study and refs. [31,34]), despite the presence of a potentially active tyrosinase [34,35], and the decrease in melanosome numbers [34,35]. Release of melanosomal contents might then impact cell viability [31]. Alternatively, it is known that melanosomes are highly enriched in divalent cations [51–53], and PMEL has been suggested to sequester calcium [54]; alterations to PMEL fibril packing might reduce its ability to sequester divalent cations, with potential harmful effects on melanosomes by further oxidative damage. Potential negative effects on copper-dependent tyrosinase activity within melanosomes might result from a similar loss of copper sequestration [55]. A third possibility is that the altered conformation of the fibrils—which very likely are a variant form of amyloid—makes them inherently toxic. For example, Ab amyloid has been shown to insert into and disrupt lipid bilayers [56]; a similar property of the TMinsWAP and TMR625C PMEL amyloid fibrils could potentially disrupt the melanosome membrane directly, leading to a loss of melanosome integrity and consequent loss of pigmentation. Finally, it is possible that pigmentation and melanosome viability are disrupted by an intermediate in fibril or sheet assembly that might persist due to a decrease in kinetics or that might be produced only by the variants. None of these possibilities are mutually exclusive, and it is possible that a combination of effects inhibits pigmentation. It has not been understood why the phenotype of the Smoky chicken, with a PKDDLVVT mutation in addition to the DW- associated TMinsWAP mutation, restores substantial pigmentation relative to the parental DW chicken [30]. We show here that the secondary PKDDLVVT mutation prevents the accumulation of PMEL in fibrillogenic compartments, likely explaining the decreased pigment dilution observed in Smoky vs. DW chickens. The PKDDLVVT mutation largely impairs PMEL maturation through the early biosynthetic pathway, causing retention in the ER. Moreover, the small fraction of PMEL that exits the ER is not selectively targeted to multivesicular endosomes and rather accumulates in early endosomal recycling compartments. This correlates with a lack of proprotein convertase cleavage into Ma and Mb fragments and decreased accumulation of PMEL fragments in detergent insoluble, fibril- enriched fractions. The behavior of this mutant form of hPMEL is similar to that of hPMEL in which the entire PKD domain is deleted [16], supporting a critical role for the PKD domain in targeting PMEL to fibrillogenic compartments and perhaps directly in fibrillogenesis [23]. The results indicate that the Smoky allele is functionally a PMEL allele that counteracts the DW mutant’s pathogenic effects on pigmentation in a recessive manner by preventing the formation of aberrant fibrils. Thus, Smoky chickens show a slight pigment dilution similar to that observed in PMEL knockout mice [29] or in the hypomorphic silver mouse [26,33] rather than a dramatic loss of eumelanin pigment as observed in the DW chicken and Silver horse. Since melanocytes in the DW chicken show decreased viability [31] and melanosome integrity [34], our studies further suggest that the formation of tightly packed TMinsWAP fibrils may be toxic to pigment cells and that it is therefore less detrimental to the cell to have no PMEL fibrils at all. The reversion of the dominant DW phenotype by the PKDDLVVT mutation in Smoky chickens suggests that a general mechanism for averting PMEL amyloid pathology is to prevent access of the aberrant amyloidogenic protein to compartments within which amyloid formation occurs. The Dun chicken appears to be another example of such a mechanism. Whereas the Dun chicken PMEL allele contains a mutation orthologous to that of the dominant TMR625C mutation in the toxic Silver horse PMEL, it also has a secondary deletion of 5 amino acids in the TMD (TMD5) [30]. Introduction of this secondary mutation into hPMEL impairs ER exit, trafficking through the plasma membrane (data not shown), and accumulation of all mature PMEL species at steady state, suggesting inefficient PMEL folding and a greater propensity for degradation. Thus, while the mechanism is different from that of the secondary PKDDLVVT mutation in Smoky chickens, the overall effect of the TMD5 mutation in Dun chickens might be similar — a reduction of aberrant PMEL accumulation in fibrillogenic compartments. We therefore liken this secondary mutation to a revertant of the Silver horse phenotype. We speculate that mutations in other toxic/ pathological forms of amyloidogenic proteins that prevent appropriate accumulation of the amyloidogenic species within amyloidogenic compartments will be associated with protection from disease. Materials and Methods Antibodies and reagents The mouse monoclonal antibodies used, their targets and sources were as follows: HMB45 and NKI-Beteb to PMEL were PLoS Genetics | www.plosgenetics.org 12 September 2011 | Volume 7 | Issue 9 | e1002286