from Lab Vision (Freemont, CA); TA99 to TYRP1 was from American Type Culture Collection (Manassas, VA); H4A3 to LAMP1 was from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). The rabbit polyclonal antibodies used, their targets and sources were aPep13h to the C-terminal peptide of hPMEL [13], aPmel-N to the N-terminal peptide of hPMEL [17], a-LAMP1 from Affinity BioReagents (Golden, CO), and a-MBP from New England Biolabs (Beverly, MA). Rat a-LAMP2 was from Developmental Studies Hybridoma Bank. Unless otherwise specified, chemicals were obtained from Sigma-Aldrich (St. Lois, MO). Tissue culture reagents were from Invitrogen (Carlsbad, CA). FuGENE-6 and hygromycin were from Roche Diagnostics (Indianapolis, IN). DNA constructs and cloning Wild-type hPMEL (long form) in pCI has been described [13] and was used as a template for site directed mutagenesis via PCR to generate the TMinsWAP, TMR625C, PKDDLVVT, PKDDLVVT- TMinsWAP, TMD5 mutations; the primers used are indicated in Table S1. Wild-type and mutant hPMEL XhoI-NotI inserts from pCI were subcloned into pBMN-IRES-Hygro (a gift from R. Scheller, Genentech, San Francisco, CA) or pBMN-IRES-EGFP retroviral vectors for stable or transient infection, respectively. For the TOXCAT assays, the pccKan expression vector was used. The pccGpA WT and pccGpA G83I mutant derivatives of pccKan, encoding wild-type or mutant glycophorin A, as well as the MBP deficient Escherichia coli (malE-) have been described [39]. PMEL wild-type or mutant TMDs were PCR-amplified from their pCI templates and cloned into the NheI-BamHI sites of pccKan. All plasmid inserts were verified by DNA sequencing. Maltose complementation and TOXCAT malE- bacteria were transformed with pccKan or its derivatives encoding the ToxR-TMD-MBP chimeras. Ampicillin resistant colonies were selected, grown to mid-log phase in Luria Broth, streaked over amp-M9 agar plates with either glucose (positive control) or maltose as the only source of carbon [39], and incubated for 2–4 days at 37uC. CAT activity of clones that grew in maltose was measured using a spectrophotometric assay [40]. Briefly, the pellet from 1 ml of OD600 = 0.75 culture was resuspended in 300 ml of sonication buffer (25 mM Tris pH 7.8 2 mM EDTA), sonicated, and centrifuged 30 min at 4uC, 13000 x g to obtain cell free extracts. 10 ml of lysate was combined with 230 ml of CAT reaction buffer (100 mM Tris pH 7.8, 0.4 mg/ml DTNB, 0.1 M AcetylCoA) and the A412 nm was recorded every minute for 5 min to obtain the background. Then, 10 ml of 2.5 mM chloramphenicol was added to initiate the CAT reaction and the A412 nm was recorded every minute for 30 min to determine the CAT activity according to the method by Shaw [40]. The change in A412 nm was linear throughout the experiment. Cell culture and transgene expression HeLa cells were grown as described previously [13] and transiently transfected with 0.1 mg DNA/ 3-cm dish for low or 7.5 mg DNA/ 10-cm dish for high transgene expression using FuGENE-6 according to the manufacturer’s instructions. HeLa cells were analyzed either 48 hrs (immunofluorescence microsco- py, metabolic labeling/pulse chase and immunoblotting) or 72– 96 hrs (electron microscopy) post-transfection. The immortalized mouse melanocyte cell lines melan-Ink4a [57] and melan- mu:MuHA rescued cells (MuHA; [58]) were grown as described previously [57] and stably or transiently infected with the retroviral vectors described above. Stable melan-Ink4a transductants were PMEL Mutations Make Functional Amyloid Pathogenic selected with 200–400 mg hygromycin B and processed for immunofluorescence (IFM) or immunoelectron microscopy (IEM). Transiently infected MuHA cells were sorted (University of Pennsylvania Cell Sorting Core Facility, Philadelphia, PA) for high EGFP expression 96 hrs post-infection and processed for conven- tional electron microscopy (EM). Immunofluorescence microscopy Cells were fixed with 2% formaldehyde, incubated with primary and fluorochrome-conjugated secondary antibodies as described previously [13] and analyzed on a DM IRBE microscope (Leica Microsystems, Wetzlar, Germany). Digital images were captured with an Orca camera (Hamamatsu, Bridgewater, NJ) and deconvolved and manipulated with OpenLab software (Improvi- sion, Lexington, MA). Insets were magnified using Adobe Photoshop (Adobe Systems, Mountain View, CA). For recycling endosome labeling by continuous transferrin (Tf) uptake, HeLa cells were starved in serum-free media containing 0.5% BSA 15 mM HEPES for 30 min at 37uC, incubated with 7.5 mg/ml Alexafluor488-conjugated human Tf (Molecular Probes, Eugene, OR) diluted in starvation media for an additional 30 min at 37uC, fixed, and processed for IFM as indicated above. Electron microscopy For conventional electron microscopy, cells were fixed in 2% glutaraldehyde 4% paraformaldehyde, dehydrated and embedded in epon resin. Ultrathin sections were contrasted with 2% uranyl acetate and analyzed by transmission electron microscopy. For immunoelectron microscopy (IEM), cells were fixed with 2% paraformaldehyde 0.5% glutaraldehyde and ultrathin frozen sections were single- or double-immunogold labeled as described previously [14,59] using Protein A gold conjugates. For quantifi- cation of numbers of Stage I-IV melanosomes, at least 50 random fields of each cell type (.400 compartments) were analyzed. Immunoblotting, metabolic labeling/pulse chase, and immunoprecipitation analyses For immunoblotting, cells were harvested with 5 mM EDTA in PBS, washed with 30 mM NEM in PBS, and frozen. Thawed cells were lysed with TX-100 lysis buffer as described previously [60] in the presence of protease inhibitors and NEM and fractionated into detergent soluble and insoluble fractions by centrifugation. For metabolic labeling, cells were harvested with trypsin-EDTA, starved for 30 min in methionine/cysteine free media, labeled with 35S methionine-cysteine for 30 min and chased for the indicated periods of time. Cells were then lysed as indicated above and detergent soluble fractions were immunoprecipitated with antibodies directed to the C-terminus of hPMEL, followed by SDS-PAGE fractionation and analysis with a STORM Phosphor- Imager and ImageQuant software (GE Healthcare, Buckingham- shire, United Kingdom). Supporting Information Figure S1 TMD mutations do not influence hPMEL covalent oligomerization and expression of TMD mutant chimeras in E. coli. A. HeLa cells transiently transfected with wild-type (wt), TMinsWAP or TMR625C variants of hPMEL were lysed, and Triton X-100 soluble fractions were fractionated by SDS-PAGE in the absence (-2ME) or presence (+2ME) of 2-mercaptoethanol. Immunoblots were probed with an antibody to the N-terminus of hPMEL. Note that the three variants migrate identically under both non-reducing and reducing conditions, indicating that each variant is capable of generating appropriate interchain disulfide PLoS Genetics | www.plosgenetics.org 13 September 2011 | Volume 7 | Issue 9 | e1002286