viability in vivo and in cell culture, and the dominant nature of the mutations, we hypothesized that the TMinsWAP and TMR625C mutations of PMEL in the DW chicken and the Silver horse, respectively, alter the cellular or biophysical properties of PMEL to ultimately convert functional amyloid into a pathological form. Here, by recapitulating these mutations in the context of human PMEL (hPMEL), we provide evidence to support this hypothesis. Moreover, we show that the secondary PKDDLVVT and TMD5 mutations in the Smoky and Dun chicken PMEL orthologues represent or partial alleles that revert the pathological effects of the TMinsWAP and TMR625C mutations on pigmentation. Finally, we show that changes in or near the TMD of an integral membrane amyloidogenic protein can influence the oligomeriza- tion of a distal lumenal fragment into functional amyloid. We discuss these findings with regard to their potential implications for the formation of functional vs. pathological amyloid. Results Mutations found in DW and HoSi PMEL alter the oligomerization potential of the human PMEL transmembrane domain Human PMEL (hPMEL) forms pre-amyloid oligomers that are stabilized by disulfide bonds [13], and a recent study found that PMEL Mutations Make Functional Amyloid Pathogenic hPMEL formed fewer disulfide-bonded oligomers when coex- pressed with chicken PMEL bearing the DW-associated TMinsWAP mutation [38]. However, it is not known whether the result in that study reflected a direct effect of the TMinsWAP mutation or an inability of hPMEL to form the appropriate disulfide bonds with chicken PMEL. To specifically test whether and how mutations in or near the PMEL TMD influence PMEL oligomerization, we introduced mutations analogous to those found in the DW chicken (TMinsWAP) and Silver horse (TMR625C) in the context of hPMEL (Figure 2A). Upon expression of these mutants or wild-type full- length hPMEL in HeLa cells, we found that the TMD mutants were equally effective as wild-type hPMEL at forming disulfide bonded oligomers, as detected by non-reducing SDS-PAGE and immunoblotting (Figure S1A). These data suggest that the PMEL mutations associated with the DW chicken and HoSi do not affect the formation of disulfide-bonded PMEL oligomers when presented within the context of hPMEL. The previously observed reduction in disulfide-bonded oligomers by hPMEL upon coex- pression of chicken DW PMEL [38] thus likely reflects the inability of these cross-species PMEL isoforms to form oligomers, which is supported by our inability to co-immunoprecipitate wild-type mouse PMEL with wild-type hPMEL (data not shown). While oligomerization of the hPMEL lumenal domain is stabilized by disulfide bonds, it is not known whether the TMD Figure 2. PMEL TMD mutations cause increased TMD–mediated dimerization. A. Wild-type human PMEL (hPMEL) TMD sequence (bold) and surrounding amino acids. In the context of hPMEL, homologous mutations to those found in the DW chicken (TMinsWAP) and Silver horse (TMR625C) are shown in gray. B. Left, TMD sequences (bold) used to make the ToxR-TMD-MBP chimeras with (L) or without (S) the N-terminal boundary residues QE and C-terminal boundary residues RRR. Right, MBP-deficient bacteria transformed with the indicated TMD-containing chimera were plated on medium with maltose as the only source of carbon; shown is whether colonies grew (+) or not (-). Growth indicates proper insertion of the chimera into the bacterial membrane. C. Representative histogram (from at least 3 similar experiments) of CAT activity, representing TMD-mediated dimerization of the chimera, as measured spectrophotometrically and normalized to protein concentration. GA, Glycophorin A TM domain. Bars, standard deviation. doi:10.1371/journal.pgen.1002286.g002 PLoS Genetics | www.plosgenetics.org 3 September 2011 | Volume 7 | Issue 9 | e1002286