bonds. B. Whole cell lysates from cultures of bacteria that were either untransformed (untrfm.) or transformed to express the indicated ToxR- PMEL-TM-MBP chimeric proteins were frac- tionated by SDS-PAGE and probed with anti-MBP antibody (IB: a- MBP). Note that all fusion proteins were expressed at similar levels. (TIF) Figure S2 Absence of fibrillar compartments in HeLa cells that do not express PMEL variants. HeLa cells transiently transfected with empty pCI vector were fixed and embedded in epon resin for conventional electron microscopy analysis, as in Figure 4. Note the absence of fibrillar structures that are observed in cells expressing either wild-type or TMD variant forms of hPMEL (Figure 4) and the presence of numerous multivesicular/ multilaminar, electron dense lysosomes (L). M, mitochondria. Bar, 500 nm. (TIF) Figure S3 Localization of wild-type, TMinsWAP and TMR625C hPMEL variants relative to lysosomes and pigment granules in melanocytes. Shown are individual panels of the overlays shown in Figure 5 for immortalized melan-Ink4a cells stably expressing wild-type (WT; top panels), TMinsWAP (middle panels) or TMR625C (bottom panels) variants of hPMEL. See legend to Figure 5 for details. Left, labeling for hPMEL variants only (green). Middle, labeling for LAMP2 only (red). Right, bright field images of pigment granules only. Insets, 4X magnifications of boxed regions, showing overlap of hPMEL variant (green) with LAMP1 (red; left panels), pigment granules (pseudocolored blue; middle panels), or with both (right panels). Bar, 10 mm. (TIF) Figure S4 PMEL TMD mutants form aberrantly packed fibrils within pigmented cells. Immortalized melan-Ink4a melanocytes that stably express wild-type (a–c), TMinsWAP (d–f ) or TMR625C (g–i) variants of hPMEL were fixed and processed for cryoimmunoelec- tron microscopy. Ultrathin cryosections were immunogold labeled for hPMEL using the hPMEL-specific NKI-beteb antibody and 10 nm protein A gold; shown are additional images to complement those in Figure 6. Note the presence of aberrantly packed, unpigmented compartments (asterisks) that are densely labeled with antibodies against hPMEL only in cells expressing the TMD mutant isoforms. Melanosomes of stage I, II, III and IV are indicated. Scale bar, 0.2 mm except in panel a, in which it represents 0.5 mm. (TIF) Figure S5 PKDDLVVT deletion found in Smoky chickens elimi- nates the formation of detergent-insoluble MaC fragments by hPMEL. HeLa cells transiently transfected with wild-type (wt), TMinsWAP, PKDDLVVT, PKDDLVVT –TMinsWAP, or TMR625C variants of hPMEL were lysed and fractionated into detergent soluble (S) and insoluble (I) fractions. Identical cell equivalents of each fraction were separated by SDS-PAGE and analyzed by immunoblotting with HMB45 anti-PMEL antibody. Left, molec- ular weight markers; right, full-length Ma and fibril-associated References 1. Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75: 333–366. 2. Greenwald J, Riek R (2010) Biology of amyloid: structure, function, and regulation. Structure 18: 1244–1260. 3. Fowler DM, Koulov AV, Balch WE, Kelly JW (2007) Functional amyloid--from bacteria to humans. Trends Biochem Sci 32: 217–224. 4. Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, et al. (2009) Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science 325: 328–332. 5. Wickner RB, Shewmaker F, Kryndushkin D, Edskes HK (2008) Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures. Bioessays 30: 955–964. PMEL Mutations Make Functional Amyloid Pathogenic MaC fragments are indicated. Note the absence of HMB45- reactive bands in cells expressing the PKDDLVVT and PKDDLVVT – TMinsWAP variants. (TIF) Figure S6 The TMD5 mutation found in Dun chickens impairs hPMEL maturation. A. Sequence of TMD residues 596-627 in wild-type and TMD5 variant hPMEL. The PMEL gene in the Dun chicken harbors an in-frame deletion that eliminates five amino acids from the middle of the TMD, in addition to the TMR625C mutation that is also found in the Silver horse PMEL. B. Immunoblot analysis. Transfected HeLa cells expressing wild-type (wt) or TMD5 hPMEL variants were lysed and fractionated into detergent soluble and insoluble fractions. Detergent soluble fractions (top three panels) were probed with antibodies to either the hPMEL N- terminus to detect P1 and Ma (top), or the C-terminus to detect Mb and CTF (middle panels). Detergent insoluble, fibril-enriched fractions were probed with HMB45 to detect the PMEL-derived MaC fragments (lower panel). Left, molecular weight markers; right, relevant bands are indicated. C. Metabolic labeling/pulse chase analysis of HeLa cells transiently transfected with wild-type or TMD5 variant hPMEL. Cells were labeled, chased and Triton X- 100-soluble cell lysates were immunoprecipitated with antibody to the hPMEL C-terminus as in Figure 3C. D. Quantification of abundance of relevant PMEL fragments from the pulse/ chase experiment shown in C.; the band intensity of each fragment was normalized to that of P1 at time zero [P1(t0)]. Note the reduced fraction of all TMD5 post-ER bands. E. IFM analysis of HeLa cells transiently expressing TMD5 variant hPMEL. Cells were labeled with NKI-beteb monoclonal antibody to PMEL (left, red) and with anti-LAMP1 antibody (overlay shown on the right). Insets show a 4X magnification of the boxed region. Note the presence of TMD5 variant hPMEL within structures circled by LAMP1, as observed for wild-type hPMEL (see Figure 3A). (TIF) Primers used to make full-length hPMEL mutants. Table S1 (DOC) Acknowledgments We thank Jeannine M. Mendrola for helpful su ggestions and for providing the pcckan and GpA vectors, Richard Scheller and Andrew Peden for the gifts of pBMN vectors, Gabriella Lindgren for informing us about the horse Silver mutation prior to publication and for helpful insights, Marc Branche for technical help with unpublished experiments, and Jim Shorter for helpful suggestions. Author Contributions Conceived and designed the experiments: BW MAL SK GR LA MSM. Performed the experiments: BW DT GR MSM. Analyzed the data: BW MAL GR LA MSM. Contributed reagents/materials/analysis tools: BW DT MAL SK GR LA MSM. Wrote the paper: BW MSM. 6. Hammer ND, Wang X, McGuffie BA, Chapman MR (2008) Amyloids: friend or foe? J Alzheimers Dis 13: 407–419. 7. Theos AC, Truschel ST, Raposo G, Marks MS (2005) The Silver locus product Pmel17/ gp100/ Silv/ ME20: Controversial in name and in function. Pigment Cell Res 18: 322–336. 8. Watt B, Raposo G, Marks MS (2010) Pmel17: An amyloid determinant of organelle structure. In :Bucciantini M, ed. Functional Amyloid Aggregation. TrivandrumKerala, , India: Research Signpost. 9. Hearing VJ (2005) Biogenesis of pigment granules: a sensitive way to regulate melanocyte function. J Dermatol Sci 37: 3–14. 10. Raposo G, Marks MS (2007) Melanosomes - dark organelles enlighten endosomal membrane transport. Nat Rev Mol Cell Biol 8: 786–798. PLoS Genetics | www.plosgenetics.org 14 September 2011 | Volume 7 | Issue 9 | e1002286