Mutations in or near the Transmembrane Domain Alter PMEL Amyloid Formation from Functional to Pathogenic Brenda Watt1,2, Danie` le Tenza3,4, Mark A. Lemmon5, Susanne Kerje6, Grac¸ a Raposo3,4, Leif Andersson6,7, Michael S. Marks1,2* 1 Department of Pathology and Laboratory Medicine and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3 Institut Curie, Centre de Recherche, Paris, France, 4 CNRS, UMR-144, Paris, France, 5 Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 6 Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, 7 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden Abstract PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)—which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core—are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show that the DW and HoSi mutations alter PMEL TMD oligomerization and/or association with membranes, with consequent formation of aberrantly packed fibrils. The aberrant fibrils are associated with a loss of pigmentation in cultured melanocytes, suggesting that they inhibit melanin production and/or melanosome integrity. A secondary mutation in the Smoky chicken, which reverts the dominant DW phenotype, prevents the accumulation of PMEL in fibrillogenic compartments and thus averts DW–associated pigment loss; a secondary mutation found in the Dun chicken likely dampens a HoSi–like dominant mutation in a similar manner. We propose that the DW and HoSi mutations alter the normally benign amyloid to a pathogenic form that antagonizes melanosome function, and that the secondary mutations found in the Smoky and Dun chickens revert or dampen pathogenicity by functioning as alleles, thus preventing the formation of aberrant fibrils. We speculate that PMEL mutations can model the conversion between physiological and pathological amyloid. Citation: Watt B, Tenza D, Lemmon MA, Kerje S, Raposo G, et al. (2011) Mutations in or near the Transmembrane Domain Alter PMEL Amyloid Formation from Functional to Pathogenic. PLoS Genet 7(9): e1002286. doi:10.1371/journal.pgen.1002286 Editor: Ian J. Jackson, Medical Research Council Human Genetics Unit, United Kingdom Received April 25, 2011; Accepted July 28, 2011; Published September 15, 2011 Copyright: 2011 Watt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the following funding sources: National Institutes of Health grant R01 AR048155 from the NIAMS (to MSM); National Institutes of Health Training Grant T32 GN997229 and Fellowship F31 GM08917 from the NIGMS (to BW); Institut Curie, CNRS, and Fondation Pour le Recherche Me´dicale (to GR); and the Swedish Foundation for Strategic Research (to LA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marksm@mail.med.upenn.edu Introduction Amyloid fibrils are polymers of single proteins that oligomerize and assemble into a characteristic fibrillar structure with a cross- beta sheet backbone [1,2]. Amyloid formation is typically associated with pathologies, such as the Ab aggregates in Alzheimer Disease or prion aggregates in inherited or acquired spongiform encephalopathies. However, the amyloid fold has also been exploited for functional means in prokaryotes, lower eukaryotes and even in mammals [2–6]. The structural and biogenetic features that distinguish functional from pathological amyloid are not well understood. Discerning these features might lead to novel therapies for amyloid diseases. A potential model for distinguishing functional from patholog- ical amyloids is the pigment cell-specific integral membrane glycoprotein, PMEL (also called gp100, Pmel17, or Silver) [7,8]. Functional amyloid fibrillar sheets, composed largely of lumenal proteolytic fragments of PMEL, form the structural foundation of eumelanosomes, which are membrane-bound, pigment cell-specific lysosome-related organelles within which black and brown melanin pigments are synthesized and stored [9,10]. The fibrils begin to form in association with intralumenal membrane vesicles (ILVs) within multivesicular melanosome precursors [11,12], to which PMEL is selectively delivered during biosynthetic transport [13,14]. Either in the trans Golgi network [15] or in association with the ILVs or with membrane domains destined for the ILVs [16], PMEL is cleaved by two site-specific proteases to liberate a lumenal fragment called Ma [13,14,16–18] (see Figure 1). Ma then undergoes ordered oligo- merization into protofibrils that are detergent insoluble [17], detectable by electron microscopy (EM) [11,13,14,17], and reactive with amyloidogenic dyes [19]. The Ma fibrils are further matured by proteolytic processing [20–23] and assemble into sheets [11,24], upon which melanins deposit as they are synthesized during melanosome maturation [10,24]. In vitro, denatured recombinant Ma fragments that are diluted into non-denaturing buffers rapidly assemble into fibrils that are classified as amyloid by a number of biophysical measures [19,23,25]. The physiological function of the fibrils is not entirely clear, but they likely serve to condense melanin PLoS Genetics | www.plosgenetics.org 1 September 2011 | Volume 7 | Issue 9 | e1002286