MFSD12 mediates the import of cysteine into melanosomes and lysosomes

Abstract

Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome—the organelle, related to the lysosome, that synthesizes pigment—but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine—the oxidized dimer of cysteine—in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7,8,9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.

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Fig. 1: MelanoIP enables the rapid isolation of pure melanosomes.
Fig. 2: MFSD12 is necessary to maintain melanosomal cystine levels and produce cysteinyldopas.
Fig. 3: MFSD12 is necessary to maintain lysosomal cystine and cysteine levels.
Fig. 4: MFSD12 is necessary and probably sufficient for the import of cysteine into melanosomes and lysosomes.

Data availability

Fig. 3a and Extended Data Fig. 3a were generated from FANTOM5 expression data, accessed via the Human Protein Atlas20,21 from https://www.proteinatlas.org/about/assays+annotation#fantom. Raw values from this accession are included in the source data for Fig. 3a and Extended Data Fig. 3a. Unique biological materials in the form of plasmids available from Addgene. Unique biological materials in the form of cell lines are available from the authors by request. Source data are provided with this paper.

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Acknowledgements

We thank P. Budde, M. Abu-Remaileh, W. W. Chen, L. Bar-Peled and J. G. Bryan, as well as all current members of the Sabatini laboratory for helpful insights. This work was supported by grants from the Leo Foundation (LF18057) and NIH (R01 CA103866, R01 CA129105 and R01 AI047389), fellowship support from the NIH (NRSA F31 CA228241-01) to C.H.A., Marshall Plan Foundation to A.K.T., HHMI XROP to B.C., and NSF (2016197106) to K.J.C. D.M.S. is an investigator of the Howard Hughes Medical Institute and an American Cancer Society Research Professor.

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Contributions

C.H.A. and D.M.S. initiated the project and designed the research plan. C.H.A. performed the experiments and analysed the data with help from A.K.T., B.C. and K.J.C. The LC–MS platform was operated by S.H.C., T.K. and C.A.L., who also had a critical role in method development for LC–MS-based assays for cysteine and cysteinyldopas. C.H.A. wrote the manuscript and D.M.S. edited it.

Corresponding author

Correspondence to David M. Sabatini.

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Peer review information Nature thanks Edmund Kunji and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 MelanoIP analysis detects changes in Tyr dependent melanosomal metabolites.

a, Schematic of melanin synthesis. The common pathway elements for eumelanin and pheomelanin synthesis have a grey backdrop. The brown and red backdrops highlight unique portions of eumelanin and pheomelanin synthesis, respectively. Enzymes proposed to catalyse each step are shown in green. Synthetic intermediates annotated and validated in biological samples in this study are in blue. b, Follow-up analysis with standard validated m/z and internal normalization of ‘proteogenic amino acids’ highlighted in untargeted metabolite profiling of wild-type and Tyr-knockout melanosomes (Fig. 1d). Amino acids are presented in order of increasing retention time (n = 3 independently prepared extracts, aP = 3.9 × 10−2, bP = 2.0 × 10−3, cP = 6.5 × 10−3, dP = 3.8 × 10−2, eP = 1.9 × 10−2). Error bars are mean ± s.e.m., P values by two-sided Student’s t-test. Source data

Extended Data Fig. 2 In vitro synthesis and biological detection of cysteinyldopas.

a, Cysteinyldopas were synthesized according to an adapted protocol from Ito and Prota, 197738. Two species, distinguished by retention time, were generated at the expected m/z for cysteinyldopas. It has been shown that 5′-cysteinyldopa is produced in greater abundance than 2’-cysteinyldopa in this reaction. Taking MS1 peak intensity to approximate abundance, we putatively annotate the ‘Minor Isomer’ as 2’ substituted, and the ‘Major Isomer’ as 5′ substituted. b, Mirror plot of ddMS2 data comparing 2’- and 5′-cysteinyldopa in synthetic cysteinyldopas. c,d, Mirror plots of ddMS2 peaks displaying similarities in ddMS2 spectra of 2’- and 5′-cysteinyldopa species in biological samples (B16F10 extracts) and synthetic standards. Source data

Extended Data Fig. 3 MFSD12 maintains lysosomal cystine in non-pigmented cells.

a, FANTOM5 CAGE profiling data accessed via Human Protein Atlas20,21. Six representative pigmentation genes, including MFSD12, are shown. b, Metabolite profiling of LysoIP samples from HEK 293T cells comparing lysosomes from wild-type and MFSD12-knockout cells. ‘Accumulates upon inhibition of:’ has been previously reported11 (n = 4 independently prepared metabolite extracts, aP = 7.0 × 10−4, bP = 3.0 × 10−3, cP = 4.1 × 10−2, dP = 4.2 × 10−2, eP = 1.7 × 10−4). c, Lentiviral shRNA knock-down of MFSD12 quantified via qPCR and normalized to ACTB levels (n = 3 assays on independently prepared cDNA libraries, aP = 1.97 × 10−3, bP = 3.0 × 10−3). Error bars are mean ± s.e.m., P values by two-sided Student’s t-test. Source data

Extended Data Fig. 4 MFSD12 mediated cysteine transport is cysteine specific.

a, Test of lysosomal counter-transport. Lysosomes were purified by differential centrifugation and incubated with water or 1 mM cysteine methyl ester before washing, resuspension, and incubated for 5 min with 20 μM cysteine and trace amounts of [35S]cysteine (n = 3 independently performed assays per condition, aP = 2.5 × 10−3, bP = 3.3 × 10−2, NS = not significant). b, Lysosomal import of [14C]cystine. Lysosomes were purified by differential centrifugation and incubated for 10 min with 1 μM [14C]cystine, either untreated (Unreduced) or pre-treated with 10 mM DTT (Reduced, n = 6 independently performed assays per condition, aP = 2.1 × 10−7, bP = 1.3 × 10−6, cP = 3.8 × 10−8). c, Competition for [35S]cysteine transport. Lysosomes were purified by differential centrifugation and incubated for 10 min with 20 μM cysteine and trace amounts of [35S]cysteine with 500 μM competitor where indicated (n = 3 independently performed assays per condition, P values compare competition condition versus water control condition (red), aP = 2.7 × 10−4, bP = 2.5 × 10−4, cP = 3.2 × 10−4). Error bars are mean ± s.e.m., P values by two-sided Student’s t-test. Source data

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Adelmann, C.H., Traunbauer, A.K., Chen, B. et al. MFSD12 mediates the import of cysteine into melanosomes and lysosomes. Nature (2020). https://doi.org/10.1038/s41586-020-2937-x

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