Diagram of PMEL domain structure, relevant proteolytic fragments and proteolytic cleavage sites (dashed lines and scissors). Within the primary sequence are indicated the signal peptide (S), N-terminal region (NTR), polycystic kidney disease repeat domain (PKD), repeat domain (RPT), transmembrane domain (TM) and cytoplasmic domain (Cyto). The signal peptide is removed from the N-terminus of PMEL in the ER by signal peptidase (SP). PMEL is cleaved by a proprotein convertase (PC) to produce Mα and Mβ fragments, which are linked by disulfide bonds (not shown). Subsequent cleavage by a site 2 protease (S2P) at a site proximal to the lumenal side of the TM produces a C-terminal fragment (CTF) and liberates Mα. As yet unidentified enzymes (marked “??”) further cleave Mα at as yet unknown sites to produce MαC. MαC fragments and a fragment of the PKD domain have been shown to accumulate in fibrillar stage II melanosomes [23].

HeLa cells were transiently transfected with low (A and C–D) or high (B) DNA levels of pCI plasmids encoding wild-type, TM^insWAP or TM^R625C hPMEL, and processed for analysis 48 hrs post-transfection. A. IFM analysis. HeLa cells were fixed and co-stained with antibodies against hPMEL (red; panels a, d and g) and LAMP1 (green; panels b, e, and h). Right panels (c, f, i) show the merged images, and insets show a 4X magnification of the boxed regions. Notice the similar pattern of labeling of wild-type (a–c), TM^insWAP (d–f) and TM^R625C hPMEL within LAMP1-labelled late endosomes/lysosomes. Bar, 20 µm. B. Immunoblot analysis. Transfected HeLa cells 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 Mα (top), or the C-terminus to detect Mβ and CTF (middle panels). Detergent insoluble, fibril-enriched fractions were probed with HMB45 to detect the PMEL-derived MαC fragments (lower panel). Left, molecular weight markers; right, relevant bands are indicated. C. Metabolic pulse chase/ immunoprecipitation analysis. Transfected HeLa cells were metabolically labeled with ³⁵S methionine/ cysteine, chased for the indicated times, and lysed in Triton X-100. PMEL was immunoprecipitated from detergent soluble fractions using an antibody to the C-terminus. Left, molecular weight markers; right, relevant bands are indicated. D. Quantification of abundance of relevant PMEL fragments from at least three pulse chase experiments as shown in C.; the band intensity of each fragment was normalized to that of P1 at time zero [P1(t₀)]. Bars, standard error.

Immortalized melan-Ink4a mouse melanocytes that stably expressed wild-type (a–c), TM^insWAP (d–f) or TM^R625C (g–i) variants of hPMEL were fixed and analyzed by IFM relative to the lysosomal marker, LAMP2 (red), and to pigment granules visualized by bright field microscopy (BF; pseudocolored blue). Ectopic hPMEL was detected using NKI-Beteb (green), an antibody that recognizes human PMEL, but not murine PMEL. Shown are overlaps of PMEL relative to LAMP2 (a, d, g) or melanosomes (b, e, h) alone or together (c, f, i). Insets show a 4X magnification of the boxed areas. Note that both mutants show a similar pattern of staining and co-localization as wild-type hPMEL. Individual labels and the original bright field images are shown in Figure S3.

Immortalized melanMu:MuHA pigmented melanocytes were transiently infected with retroviruses that encode wild-type, TM^insWAP or TM^R625C hPMEL-and co-expressed EGFP. Four days post-infection, cells expressing high levels of hPMEL transgene were selected by flow cytometric sorting for high EGFP expression, then fixed, embedded in epon resin, and processed for conventional thin section EM. A. Cells expressing wild-type hPMEL (left panels) show predominantly pigmented stage III and IV (III, IV) melanosomes and few unpigmented Stage I and II melanosomes (I, II). By contrast, cells expressing either TM^insWAP (middle panels) or TM^R625C (right panels) hPMEL variants harbor many fewer Stage IV melanosomes and many more non-pigmented, Stage I-II melanosomes. M, mitochondria. B. The total number of melanosomes of each stage in 6–7 whole cells from each set of samples was quantified relative to the total number of melanosomes per cell. The mean, median, maximum (max) and minimum (min) values, and 25th and 75th quartile values for distance moved in each experimental set are shown. C. Quantification of the number of each stage melanosome divided by the number of melanosomes in that field; at least 60 fields of equal size/magnification were analyzed, containing more than 400 melanosomes of different maturation stages. *, p <0.05; **, p <0.01 as determined by ANOVA and Student's t-test. D. Quantification of the number of pigmented granules (Stage III-IV) per µm² of cell area in cells expressing wild-type, TM^insWAP or TM^R625C hPMEL variants or cells transduced with virus that did not co-express hPMEL. Each measurement is shown individually; median values are indicated. **, p<0.01 as determined by ANOVA and Student's t-test.

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 (TM^insWAP) and Silver horse (TM^R625C) 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.

HeLa cells transiently overexpressing wild-type, TM^insWAP or TM^R625C hPMEL were fixed and embedded in epon resin for conventional electron microscopy analysis. Note that elongated fibrils in the lumen of multivesicular endocytic compartments (labeled with asterisks) are observed in cells expressing all three variants of hPMEL. Bars, 500 nm.

Immortalized melan-Ink4a melanocytes that stably express wild-type (a, d), TM^insWAP (b, e) or TM^R625C (c, f) variants of hPMEL were fixed and processed for cryoimmunoelectron microscopy. Ultrathin cryosections were immunogold labeled for hPMEL using the hPMEL-specific NKI-beteb antibody and 10 nm protein A gold. Note that dense immunolabeling is observed on short immature fibrils (arrowheads) within stage I melanosomes from cells expressing all three hPMEL variants (upper panels, a–c), but that mature, elongated fibrils are immunogold labeled only in compartments (asterisks) within cells expressing the TMD mutants (lower panels, d–f). Also note the densely packed profile of the immunogold-labeled mature fibrils in cells expressing TMD mutant hPMEL as compared to the thick pigmented fibrils that lack labeling, which are found within stage III melanosomes in cells expressing wild-type hPMEL and which show some spacing between neighboring fibrils (bracket). Bars, 200 nm.

A. Sequence of PKD domain residues 245–280 in wild-type and mutant hPMEL. The PMEL gene in the Smoky chicken harbors an in-frame deletion that eliminates four amino acids from the PKD domain, corresponding to LVVT in hPMEL (PKD^ΔLLVT), in addition to the TM^insWAP mutation. B. Metabolic labeling/pulse chase analysis of HeLa cells transiently transfected with wild-type, PKD^ΔLVVT, TM^insWAP or PKD^ΔLVVT-TM^insWAP variants of hPMEL. Cells were labeled, chased and Triton X-100-soluble cell lysates were immunoprecipitated with an antibody to the hPMEL C-terminus as in Figure 3C. Note that variants bearing the PKD^ΔLVVT deletion, regardless of the presence of the TM^insWAP insertion, do not mature efficiently to P2 and fail to accumulate Mα and Mβ fragments. C. IFM analysis of HeLa cells transiently expressing wild-type (a–c), PKD^ΔLVVT (d–f) or PKD^ΔLVVT-TM^insWAP (g–i) variants of hPMEL. Cells were fixed after exposure for 30 min at 37°C to fluorophore-conjugated transferrin (Tf) to label early sorting and recycling endosomes, then stained with an antibody that recognizes only mature PMEL (HMB45) and an anti-LAMP1 antibody. Left panels (a, d, g) show PMEL labeling only; middle panels (b, e, h) show overlap of LAMP1 (green) and HMB45-labeled PMEL (pseudocolored red); and right panels (c, f, i) show overlap of internalized Tf (green) and PMEL (red). Insets show a 4X magnification of the boxed areas. Note that wild-type PMEL overlaps with LAMP1 but not internalized Tf, whereas PKD^ΔLLVT overlaps partially with Tf but not LAMP1.