Domain mapping and processing of Pmel17. A) Pmel17-i is subdivided into the following 10 domains: SIG, signal peptide; NTD, N-terminal domain; PKD, polycystic kidney disease-like domain; GAP1, undefined domain between PKD and RPT; RPT, proline, serine, threonine-rich repeat domain; GAP2, undefined domain between RPT and KRG; KRG, kringle-like domain; GAP3, undefined domain between KRG and TM; TM, transmembrane domain; CTD, C-terminal domain. Numbers represent amino acid count based on Pmel17-i. Five potential N-glycosylation sites are indicated with circles; however, one is not likely used (open circle). Furin-mediated cleavage site (CS) in GAP2 is shown with a dashed line and is subdivided into GAP2a and GAP2b by the CS. RPT domain is further subdivided into 10 imperfect series of 13 aa each. Each series is annotated from a to j. Asterisks indicate potential O-glycosylation sites. Four isoforms of Pmel17 are currently identified. RPT domains of Pmel17-is and Pmel17-ls lack the underscored 42 aa. Pmel17-l and Pmel17-ls contain the underscored 7 aa in GAP3. B) Schematics of the processing of Pmel17. P1/P100 is the partially glycosylated form of Pmel17. P2/P120 is the fully glycosylated form of Pmel17; check marks indicate mature N-linked glycans. Mα and Mβ/P26 are the cleaved products P2/P120, which may be disulfide-bond linked. Mα is also believed to be the secreted form of Pmel17 (sPmel17). Scheme at bottom shows the further processed Pmel17 forms found in stage II melanosomes. Mα is further cleaved into MαN and MαC, then MαC is subject to multiple shedding. Mβ is also further cleaved at S2 by the metalloproteinases and at γ by γ-secretases. αPEP13h reacts with the carboxyl terminus of Pmel17. HMB50 and NKI/beteb are believed to react with the lumenal domain, but the precise epitopes recognized by HMB50 or NKI/beteb are unknown. HMB45 has been proven to react with sialylated RPT domain. This schematic is based on previous publications (10, 11, 14, 15, 17, 19, 272829, 34). Nomenclatures P1/P100, P2/P120, Mα, Mβ/P26, MαN, and MαC were taken from refs. 10, 14, 34.

NTD, PKD, GAP1, and GAP2b domains are required for the immunoreactivity of HMB50 or NKI/beteb. EGFP constructs used in this study are shown. EGFP-NP12 contains EGFP and the NTD, PKD, GAP1, and GAP2 domains. EGFP-NP12a contains EGFP and the NTD, PKD, GAP1, and GAP2a domains. EGFP-NP12b contains EGFP and the NTD, PKD, GAP1, and GAP2b domains. EGFP-N_196–254P12b contains part of the NTD (aa 196–254), PKD, GAP1, and GAP2b domains. HeLa cells overexpressing EGFP-NP12, EGFP-NP12a, EGFP-NP12b, and EGFP-N_196–254P12b were fixed and stained with HMB50 or NKI/beteb and then were reacted with Alexa Fluor 594 or 647, respectively. Scale bars = 20 μm.

Pmel17 secretion is independent from the cleavage by PCs. A) HeLa cells overexpressing Pmel17-i, Pmel17-l, Pmel17-i-ΔCS, or Pmel17-l-ΔCS were radiolabeled, then chased for 3 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, separated by electrophoresis, and visualized by autoradiography. Film was developed after a relatively long exposure time to evaluate faster-migrating bands in the medium of Pmel17-i and Pmel17-l (bottom panel). B) MNT-1 cells with or without dec-RVKR-CMK at 50 μM were radiolabeled, then chased for 3 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, separated by electrophoresis, and visualized by autoradiography. MNT-1 cells transduced with adenovirus containing LacZ (control) or α1-PDX (engineered mutant of α1-antitrypsin) were radiolabeled and chased for 3 h, then analyzed as described previously (top panel). LoVo cells transduced with adenovirus containing Pmel17-i or Pmel17-i + furin were radiolabeled and chased for 3 h, then analyzed as described previously (bottom panel). C) MNT-1 cells and HeLa cells overexpressing Pmel17-i were radiolabeled and chased for 3 h. Cell lysates and medium were immunoprecipitated with NKI/beteb. Immunopurified samples were incubated with or without 2ME, then separated by electrophoresis and visualized by autoradiography.

Secreted Pmel17 is fully Golgi modified, and the N-glycan in GAP3 contributes to the secretion. A) MNT-1 cells and HeLa cells overexpressing Pmel17-i were radiolabeled and chased for 1 h (cell lysates of MNT-1 cells) or 4 h (HeLa cells and medium of MNT-1 cells). Cell lysates and medium were immunoprecipitated with NKI/beteb. Immunopurified samples were split into 3 portions; one was digested with EndoH (750 U), another was digested with PNGaseF (750 U), and the third was the untreated control. Digestion reactions were performed for 12 h at 37°C. Digested samples were electrophoresed and visualized by autoradiography. B) MNT-1 cells were radiolabeled and chased in the presence of DMSO, Tun at 2 μg/ml, or DMM at 1 mM for 4 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, separated by electrophoresis, and visualized by autoradiography. Intensities of specific bands, Mα, relative to control are quantified (bottom panel). Graphed data are mean ± sd ratios of Mα intensity in medium/cells from 3 independent experiments. C) HeLa cells overexpressing S83A, T108A, S113A, S83A/T108A/S113A, or S570A were radiolabeled and then analyzed as described above.

Schematic of HMB50 and NKI/beteb epitopes and the secreted form of Pmel17 (sPmel17). HMB50 and NKI/beteb react with parts of NTD, PKD, GAP1, and GAP2b (indicated by horizontal line). Some of the Mα-Mβ complex is further processed in premelanosomes or in melanosomes (Fig. 1), and some is further cleaved into the secreted form, Mα-MβN, probably by multiple ectodomain shedding at spanning or juxtamembrane domains. However, the exact cleavage points are currently unknown (indicated by question mark). Remainder of Mβ is annotated as MβC. Mα and MβN remain linked in the medium. Checkmark indicates a fully matured N-glycan; ν indicates a high-mannose-type or hybrid-type N-glycan.

Mutants of Pmel17 analyzed with HMB50 and NKI/beteb. A) HeLa cells transfected with mutants of Pmel17, including ΔSIG (SIG domain is artificially deleted), ΔNTD, ΔPKD, ΔGAP1, ΔRPT, ΔGAP2a, ΔGAP2b, ΔKRG, ΔGAP3, ΔTM, and ΔCTD were immunocytochemically stained with HMB50, HMB45, and NKI/beteb and then reacted with Alexa Fluor 594, 488, and 647, respectively. Scale bars = 20 μm. B) HeLa cells transfected with mutants of Pmel17, including ΔSIG, ΔNTD, ΔPKD, ΔGAP1, ΔRPT, ΔGAP2, ΔGAP2a, ΔGAP2b, ΔKRG, ΔGAP3, ΔTM, and ΔCTD, analyzed by immunoblotting using αPEP13h.

sPmel17 consists of Mα and lower-molecular-weight fragment, MβN. A) Highly pigmented MNT-1 cells were metabolically radiolabeled, then chased for specific periods as noted. Cell lysates and medium were immunoprecipitated with HMB50 or NKI/beteb, separated by electrophoresis, and visualized by autoradiography. Faster-migrating bands in the medium are annotated as MβN. Asterisk indicates the nonspecific band. B) NHMs were radiolabeled and chased as described above. C) HeLa cells overexpressing an empty vector, Pmel17-i, Pmel17-l, Pmel17-is, or Pmel17-ls were radiolabeled, then chased for 3 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, separated by electrophoresis, and visualized by autoradiography.

ΔKRG or ΔGAP3 does not abrogate the secretion of Pmel17. A) HeLa cells overexpressing mutants of Pmel17, including ΔKRG, ΔGAP3, ΔTM, and ΔCTD, were radiolabeled and chased for 4 h. Cell lysates and medium were immunoprecipitated with NKI/beteb and then analyzed as described previously. B) HeLa cells overexpressing Pmel17-i and ΔGAP3 were radiolabeled and chased for 4 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, then analyzed as described previously. Note that film was developed after a relatively long exposure time to analyze faster-migrating bands in the medium of Pmel17-i and Pmel17-ΔGAP3. C) HeLa cells overexpressing Pmel17-i and ΔS2 were radiolabeled and chased for 4 h; cell lysates and medium were immunoprecipitated with NKI/beteb, then analyzed as described previously.

Secretion of Pmel17 is up-regulated by PMA, NaN₃, or W-7, but is not inhibited by metal chelators. A) MNT-1 cells were immunocytochemically stained with HMB50 in the presence or absence of TX. Scale bar = 20 μm. B–D) MNT-1 cells were radiolabeled and chased in the presence of 200 nM PMA, DMSO, 20 mM NaN₃, 5 mM EGTA, and 5 mM EDTA (B), 50 μM W-7 (C), or 100 nM GM-6001 and 10 μg/ml BFA (D) for 4 h. One sample was chased at 4°C. Medium was analyzed as described previously. Intensities of specific bands, Mα, relative to control are quantified (bottom panel). Graphed data are means ± sd from 3 independent experiments.