itself has oligomeric properties that might otherwise influence lumenal domain interactions. To determine whether the hPMEL TMD can oligomerize on its own and whether the TMinsWAP and TMR625C mutations influence this property, we turned to the widely used TOXCAT assay [39]. In this assay, a chimeric protein consisting of the transcription factor ToxR, the TMD of interest, and maltose binding protein (MBP) is expressed in MBP-deficient bacteria. Proper insertion of the chimeric protein into the plasma membrane confers growth in maltose as the only carbon source, and oligomerization mediated by the TMD activates ToxR and stimulates ToxR-dependent transcription of the chloramphenicol acetyl transferase (CAT) gene. Upon expression of ToxR-TMD- MBP chimeras containing the PMEL TMD with or without the natural border residues at either end (Figure 2B), only chimeras lacking the C-terminal border residues, RRR (SS, LS), conferred growth in maltose — regardless of the presence of the N-terminal border residues, QE — despite equivalent expression of all chimeras as determined by immunoblotting (Figure S1B). This suggested that the C-terminal residues interfered with insertion of the chimera into the plasma membrane with the proper orientation. Interestingly, altering the second arginine in the C- terminal border sequence to either Cys (R/C; as in the TMR625C mutant), Ser (R/S), or Lys (R/K) allowed the chimeric protein to insert properly into the membrane, as evidenced by maltose complementation (Figure 2B). This indicates that the TMR625C substitution associated with HoSi PMEL alters the properties of the PMEL TMD. For those chimeras that confer growth in maltose, we then tested their ability to dimerize by measuring CAT activity using a spectrophotometric assay (Figure 2C) [40]. As a positive control we used a chimera containing the strongly dimerizing TMD of Glycophorin A (GA wt), and as a negative control we used either the vector without a TMD (no TM) or a chimera containing the non-dimerizing Glycophorin A G83I mutant TMD (GA mut) [39,41]. The wild-type PMEL TMD, lacking (SS) or containing (LS) the N-terminal border residues, conferred similar CAT activity as the negative controls. In contrast, the TMinsWAP (InsWAP) and TMR625C (R/C) mutations conferred substantial CAT activity. These data indicate that whereas the wild-type hPMEL TMD is not capable of oligomerization, both the DW- associated TMD insertion and the HoSi-associated R/C transver- sion facilitate TMD dimerization. Insertion of three leucines (InsLLL) in place of the WAP insertion conferred an even slightly higher CAT activity, suggesting that the increased dimerization mediated by the TMinsWAP mutation is likely due to the increase in TMD length. To test whether the R/C mutation reflected a specific property of the cysteine (such as disulfide bond formation), we tested whether replacement of the same arginine residue by serine (R/S) or lysine (R/K) affected CAT activity. Interestingly, whereas both of these mutants conferred proper insertion into the plasma membrane as indicated by growth in maltose, the R/S mutant, but not R/K, conferred CAT activity similar to that by the R/C mutant. This suggests that the dimerization conferred by the R/C mutant reflects decreased repulsion between adjacent basic RRR (or RKR) motifs. DW– and HoSi–associated TMD mutations do not affect PMEL trafficking to premelanosomal compartments or proteolytic activation We next tested whether the altered TMD properties associated with the TMinsWAP and TMR625C mutations influenced PMEL trafficking or processing. hPMEL without (wild-type) or with the TMinsWAP or TMR625C mutations was expressed ectopically in non-melanocytic HeLa cells by transient transfection. As previously PMEL Mutations Make Functional Amyloid Pathogenic shown, wild-type hPMEL expressed in these cells is enriched at steady state within late endosomes and lysosomes [13] (Figure 3Aa– c). Neither the TMinsWAP nor the TMR625C mutation affected this steady state localization, as shown by the predominant labeling for these mutants by immunofluorescence microscopy (IFM) on the interior of structures labeled by the late endosome/lysosome marker, LAMP1 (Figure 3Ad–i). These data suggest that the TMD mutations in dominant PMEL mutants do not affect hPMEL trafficking, supporting a similar earlier conclusion for chicken DW PMEL [38]. In order to liberate the amyloidogenic Ma fragment, PMEL undergoes regulated proteolytic cleavage in the lumenal domain by a proprotein convertase (PC) and an as yet unidentified site 2 protease (S2P) [17,18] (Figure 1). Ma is then further processed by as yet unidentified proteases during fibril maturation [12,21–23]. The products of these cleavages are detected and semi-quantified by immunoblot analysis of detergent-soluble lysates from HeLa cells transfected with wild-type hPMEL; the characteristic Ma and Mb fragments result from PC cleavage of full-length hPMEL [17], the C-terminal fragment (CTF) results from S2P cleavage of Mb [18], and the MaC fragments that are enriched in detergent- insoluble fibrils result from further proteolysis of Ma [21] (Figure 1). Similar levels of all fragments are present in lysates from cells expressing TMinsWAP or TMR625C hPMEL, with the exception of a slight increase in the levels of the CTF relative to Mb in cells expressing the TMR625C mutant (Figure 3B). Moreover, whereas wild-type and TMinsWAP hPMEL often form two species of CTF (see doublet in Figure 3B), TMR625C hPMEL forms predominantly a single species. These data suggest that TMinsWAP and TMR625C hPMEL are effectively cleaved to fibrillogenic fragments by the PC and by proteases within late endosomal compartments but that S2P cleavage is favored at one of two sites in TMR625C hPMEL and the ensuing CTF is likely more stable than the wild-type CTF. To test whether processing kinetics are altered by the TMD mutations, we analyzed PMEL maturation in transfected HeLa cells by metabolic pulse/ chase analysis of wild-type, TMinsWAP, or TMR625C hPMEL immunoprecipitated from detergent-soluble lysates. As shown in Figure 3C, both the TMinsWAP and TMR625C mutant hPMEL were matured to the Golgi-processed P2 form, cleaved to the Ma/ Mb forms, and disappeared from detergent- soluble lysates with roughly wild-type kinetics (Figure 3D). Moreover, for all hPMEL variants, Ma was secreted into the medium with similar kinetics and efficiency, and CTF was generated with similar kinetics (data not shown). Altogether, these results indicate that the TMD mutations found in the DW chicken and the Silver horse affect neither the delivery of PMEL to late endocytic compartments nor its ability to be processed to amyloidogenic Ma and MaC fragments. hPMEL with TMinsWAP and TMR625C mutations is fibrillogenic within endosomes PMEL fibril formation is often inferred from the detection of detergent-insoluble MaC fragments by immunoblotting, but non- fibrillar mutants of PMEL that form disorganized aggregates — such as hPMEL lacking a PC cleavage site — can also generate similar fragments [12,17]. To test whether the TMinsWAP and TMR625C variants are capable of supporting fibril formation, HeLa cells transiently expressing these variants or wild-type hPMEL were analyzed by standard electron microscopy (EM). As shown in Figure 4, stage II-melanosome-like compartments with fibrillar arrays were detected in cells expressing any of the hPMEL variants, but not in cells expressing empty vector (Figure S2). As for cells expressing wild-type hPMEL, fibrils in cells expressing PLoS Genetics | www.plosgenetics.org 4 September 2011 | Volume 7 | Issue 9 | e1002286