Formation of Pmel17 Amyloid Is Regulated byJuxtamembrane Metalloproteinase Cleavage, and theResulting C-terminal Fragment Is a Substrate for �-Secretase*

Received for publication, November 24, 2008 Published, JBC Papers in Press, December 1, 2008, DOI 10.1074/jbc.M808904200

Markus P. Kummer‡§, Hiroko Maruyama‡, Claudia Huelsmann§, Sandra Baches¶, Sascha Weggen¶,and Edward H. Koo‡1

From the ‡Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, the ¶MolecularNeuropathology Group, Department of Neuropathology, Heinrich Heine-University, D-40225 Duesseldorf, Germany, and the§Clinical Neurosciences Group, Department of Neurology, Rheinische Friedrich Wilhelms-University, D-53127 Bonn, Germany

The formation of insoluble cross�-sheet amyloid is patholog-ically associated with disorders such as Alzheimer, Parkinson,and Huntington diseases. One exception is the nonpathologicalamyloid derived from the protein Pmel17 within melanosomesto generate melanin pigment. Here we show that the formationof insoluble M�C intracellular fragments of Pmel17, which arethe direct precursors to Pmel17 amyloid, depends on a noveljuxtamembrane cleavage at amino acid position 583 betweenthe furin-like proprotein convertase cleavage site and the trans-membrane domain. The resulting Pmel17 C-terminal fragmentis then processed by the �-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to theNotch receptor, we designate this cleavage the S2 cleavage site,whereas �-secretasemediates proteolysis at the intramembraneS3 site. Substitutions or deletions at this S2 cleavage site, the useof themetalloproteinase inhibitorTAPI-2, aswell as small inter-fering RNA-mediated knock-down of the metalloproteinasesADAM10 and 17 reduced the formation of insoluble Pmel17fragments. These results demonstrate that the release of thePmel17 ectodomain, which is critical for melanin amyloidogen-esis, is initiated by S2 cleavage at a juxtamembrane position.

Folding of proteins is a highly regulated process ensuringtheir correct three-dimensional structure. Under pathologicalcircumstances, a soluble protein can be folded into highly stablecross �-sheet amyloid structures, which are believed to playpathological roles in disorders such as Alzheimer, Parkinson,and Huntington diseases. An exception to this general conceptis the physiological amyloid structure of the melanosomalmatrix formed by the protein Pmel17. Melanosomes are lyso-some-related organelles that contain pigment granules (mela-nin) in melanocytes and retinal epithelial cells (reviewed in Ref.1). Melanogenesis is believed to proceed through several

sequential maturation steps, classified by melanosomes fromstage I to stage IV.Maturation of stage IImelanosomes requiresthe formation of Pmel17 intralumenal fibers (2, 3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I

transmembrane glycoprotein of up to 668 amino acids inhumans (reviewed inRef. 4). The requirement of Pmel17 for thegeneration of functional melanin has been shown in a numberof different organisms, because, for example, certain pointmutations in the Pmel17/silver gene result in hypopigmenta-tion phenotypes (5–7). The most characteristic domain withinPmel17 is a specific lumenal proline/serine/threonine richrepeat domain (see Fig. 1A), that is imperfectly repeated 13times in the M� fragment. Importantly, deletion of the richrepeat domain results in a complete loss of fibril formation,pointing to the requirement of Pmel17, and especially the richrepeat domain, in melanin formation (8). Pmel17 exists in dif-ferent isoforms generated by alternative splicing. Pmel17-i2 isthemost abundant isoform, whereas the Pmel17-l isoform con-tains a 7-amino acid insertion close to the transmembranedomain (9, 10).Pmel17 traffics through the secretory pathway as a 100-kDa

protein (called P1). In the late Golgi compartment it undergoesfurther glycosylation, resulting in a short lived 120-kDa protein(called P2). P2 is rapidly cleaved within the post-Golgi by afurin-like proprotein convertase (PC) to generate two frag-ments that remain tethered to each other by disulfide bonds: aC-terminal polypeptide containing the transmembranedomain (M�) and a largeN-terminal ectodomain (M�) (2) (Fig.1A). Consequently, inhibition of this furin-like activity not onlyprevents the generation of M� and M� fragments but alsoinhibits the formation of melanosomal striation in HeLa cells

* This work was supported, in whole or in part, by National Institutes of HealthGrant AG12376 (to E. H. K.). This work was also supported by DeutscheForschungsgemeinschaft Emmy Noether Program Grant WE 2561/1-3 (toS. W.). The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

1 To whom correspondence should be addressed: Dept. of Neurosciences,University of California San Diego, La Jolla, CA 92093. Tel.: 49-858-8221024;Fax: 49-858-8221021; E-mail: edkoo@ucsd.edu.

2 The abbreviations used are: Pmel17-i, intermediate form of Pmel17,Pmel17-l, long form of Pmel17; Pmel17-CTF, Pmel17 C-terminal fragment;Pmel17-ICD, Pmel17 intracellular domain; M�, Pmel17-derived ectodo-main; M�C, C-terminal fragments of M�; DAPT, N-[N-(3,5-difluorophenac-etyl-L-alanyl)]-S-phenylglycine t-butyl ester; TAPI-2, N-(R)-(2-(hydroxyami-nocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-glycine-L-alanine-2-aminoethyl amide; PMA, phorbol 12-myristate 13-acetate; Dec-RVKR-CMK,decanoyl-Arg-Val-Lys-Arg-chloromethylketone; PC, proprotein conver-tase; siRNA, small interfering RNA; M�, C-terminal polypeptide containingthe transmembrane domain; PS, presenilin; APP, amyloid precursor pro-tein; DMEM, Dulbecco’s modified Eagle’s medium; BisTris, 2-[bis(2-hy-droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hy-droxy-1,1-bis(hydroxymethyl)ethyl]glycine; CTF, C-terminal fragment;MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 4, pp. 2296 –2306, January 23, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(3). These findings suggest that M� must first be dissociatedfrom the M� for melanogenesis to proceed. It is unclear howM� is released from the membrane. Reduction of disulfidebonds would release M� from M�; alternatively, proteolyticdigestion of M� should also free M� from the membranetether. It has been speculated that, given the presence of lyso-somal hydrolases in melanosomes and proteolytic maturationof Pmel17, proteolysis is the more likely mechanism (4).Recently, it was shown that recombinant M� is able to formamyloid structures in vitro in an unprecedented rapidity, andfurthermore, Pmel17 amyloid also accelerated melanin forma-tion (11). These findings demonstrate thatmammalian amyloidformed by Pmel17 is functional and physiological.The insoluble pool of Pmel17 in cells consists mostly of trun-

catedM�C-terminal fragments (M�C) of heterogeneous sizes,indicating that further processing ofM� occurs after its releasefrom the membrane (8, 12). M�C fragments are found in theinsoluble fraction of melanocytes as well as in nonmelanoticcells, the latter after overexpression of Pmel17 (8), and arereduced or absent in amelanotic cells (8, 13, 14).Meanwhile, theC-terminal fragment derived from theM� fragment and recog-nized by a C-terminal specific epitope antibody is less stable,indicating rapid turnover (2).The presenilin (PS) family of proteins consists of two homol-

ogous integral transmembrane proteins, PS1 and PS2, whichare part of the �-secretase complex. The latter consists of pre-senilin 1 or 2, nicastrin, APH-1, and PEN-2 (15) and catalyzesthe cleavage of the hydrophobic transmembrane domain of aburgeoning list of proteins, also called regulated intramem-brane cleavage. Other substrates for the �-secretase-mediatedintramembrane cleavage include Notch, amyloid precursorprotein (APP), cadherin (E-cadherin), nectin-1, the low densitylipoprotein-related receptor, CD44, ErbB-4, the voltage-gatedsodium channel �2-subunit, and the Notch ligands Delta andJagged. Importantly, in Alzheimer disease, the presenilin-me-diated �-secretase cleavage of APP releases the amyloid �-pro-tein fragment, a peptide believed to play a key role in Alzheimerdisease pathogenesis. Interestingly, a recent report describedthe absence ofmelanin pigment in presenilin-deficient animals,an observation confirmed by the lack of melanin formation incells treated with �-secretase inhibitors (16). The mechanismresponsible for this finding is unclear, leading us to ask whetherPmel17 processing is a presenilin-dependent process and, if so,whether this cleavage is involved in melanogenesis.In this study, we show the presence of an endoproteolytic

activity that cleaves the extracellular domain of Pmel17-i at ajuxtamembrane position between the known PC cleavage siteand the transmembrane domain,whichwe term the S2 cleavagesite, by a TAPI-sensitive ADAM (a disintegrin and metallopro-teinase protein) protease. This intracellular shedding of Pmel17after S2 cleavage results in the liberation of the M� N-terminalectodomain, the precursor to Pmel17 amyloid, which is able toform insoluble Pmel17 aggregates. The C-terminal transmem-brane fragment generated by S2 cleavage is further processedby �-secretase (S3 cleavage) to release the Pmel17 intracellulardomain, which is then rapidly degraded.

EXPERIMENTAL PROCEDURES

Drugs and Antibodies—The �-secretase inhibitors N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), the protein kinaseC activator phorbol-12-myris-tate-13-acetate (PMA), and the furin inhibitor Dec-RVKR-CMK were purchased from EMB Biosciences (San Diego, CA).TAPI-2was purchased fromBiomol (Hamburg,Germany). Theaffinity-purified polyclonal antibody �Pep13h against theC-terminus of human Pmel17 was kindly provided by Dr. M.Marks. The tubulin antibody E7 was obtained from the Devel-opmental Studies Hybridoma Bank (Iowa City, IA). Commer-cial antibodies include HMB45 (Dako, Carpinteria, CA), nicas-trin N1660 (Sigma), ADAM10 54012 (AnaSpec, San Diego,CA), ADAM17 PC491 (Calbiochem, San Diego, CA) and thec-Myc (A-14) antibody (Santa Cruz Biotechnology, Santa Cruz,CA).Cell Culture—Murine B16-F0 melanoma cells, human HeLa

cells, and murine presenilin-deficient fibroblasts were main-tained in DMEM supplemented with 10% fetal bovine serum,and 100 unit ml�1 penicillin/streptomycin. MNT-1 humanmelanoma cells (a kind gift of Dr. M. Marks) were main-tained in DMEM supplemented with 20% fetal calf serum,10% AIM-V medium (Invitrogen), and 100 units ml�1

penicillin/streptomycin.cDNA Constructs—The plasmid pCI-Pmel17-l encoding the

long form of human Pmel17 has been described previously (2)and was kindly provided by Dr. Michael Marks. The pCI-Pmel17-l-Myc was generated by introducing the Myc tagepitope at the C terminus of Pmel17 using pCI-Pmel17-l as atemplate. The intermediate isoform of Pmel17, pCI-Pmel17-i-Myc, lacking the juxtamembrane 7-amino acid insertion, aswell as all other Pmel17 mutants were generated using theQuikChange mutagenesis kit (Stratagene, La Jolla, CA).Transfections—Plasmids were transfected using Lipo-

fectamine 2000 (Invitrogen) or FuGENE (Roche Applied Sci-ence) according to the manufacturer’s recommendations. Fornicastrin RNA interference experiments, the cells weretransfected twice on two consecutive days with 100 pmol ofsiRNA duplexes (17) using Lipofectamine 2000 according tothe manufacturer’s instructions. For siRNA knock-down ofADAM proteases, the cells were cotransfected with 1 �g ofPmel17-i and 62.5 pmol of control siRNA (Qiagen; 1027292),ADAM10 siRNA pool (GUCUGUUAUUGAUGGAA-GAdTdT, CUGUGCAGAUCAUUCAGUAdTdT, and CUU-ACAAUGUGGAUUCAUUdTdT; Invitrogen; 1220067,121220, and 1212209), or ADAM17 validated siRNA AAG-AAACAGAGTGCTAATTTA (Qiagen; SI02664508) usingLipofectamine 2000 (Invitrogen). The cells were harvested48 h after transfection.Immunoblotting—The cell were lysed in 25mMTris-HCl, pH

7.5, 150 mM NaCl, 1% Triton X-100, and complete proteaseinhibitor mixture (Roche Applied Science) for 30 min on ice,and the lysateswere cleared by centrifugation at 11,000 rpm.Toextract the insoluble pool, the remaining pellets were homoge-nized in 2% SDS, 25 mM Tris-HCl, pH 7.5, by sonication. Theproteins were separated by BisTris or Tricine-SDS-PAGE andelectroblotted onto 0.45 �M polyvinylidene difluoride mem-

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branes (Millipore, Billerica, MA). For size determination theprestained molecular mass markers were used until otherwisenoted; therefore theirmigration is only approximate. Followingblocking in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% (w/v)nonfat dry milk and 0.05% Tween 20, the membranes wereincubated sequentially with the indicated primary antibody andhorseradish peroxidase-conjugated secondary antibodies. Thesignals were detected by enhanced chemiluminescence (Pierce)and quantified using a CCD camera-based imaging system(GeneGnome; Syngene, Frederick,MD). All of the experimentswere repeated two to three times, and either the results areexpressed as averages of all of the experiments � S.E. or a rep-resentative experiment is shown.Matrix-assisted Laser Desorption/Ionization Time-of-Flight

Mass Spectrometry—MNT-1 cells were treated with the�-secretase inhibitor DAPT for 3 days. Pmel17 was immuno-precipitated from Triton-solubilized cell lysates with �Pep13hantibody. The resultant samples were eluted with water:aceto-nitril:trifluoro acetic acid 50:50:0.5 (v/v/v) and mixed 1:1 (v/v)with a saturated solution of sinapinic acid in the same solventprior to spotting on a gold chip for mass spectrometry with aCiphergen SELDI system (Ciphergen Biosystems, Fremont,CA). Calibration was done using an external peptide standard.Radiosequencing—HEK293T cells were transfected with

pCI-Pmel17-i-Myc using FuGENE reagent. Two days later, thecells were starved for 30 min in methionine-free DMEM andafterward incubated in methionine free DMEM containing 1mCi of [35S]methionine, 5 �M DAPT, 1% dialyzed fetal calfserum, and 25 mM HEPES, pH 7.2, for 5 h. The cells were lysedin 1.5 ml of lysis buffer (25 mMTris-HCl, pH 7.5, 150mMNaCl,1% Triton X-100, and complete protease inhibitor mixture),and the supernatant was precipitated using the c-Myc (A-14)antibody. The precipitates were separated on a 4–12% Nupage(Invitrogen) gel, which had been equilibrated for 30min at 50 Vin the presence of 5 mM thioglycolic acid in the cathode bufferand then transferred onto Immobilon-PSQ (Millipore, Bed-ford, MA). The radiolabeled Pmel17-C-terminal fragment(CTF) band was excised from the membrane after overnightexposure to film and analyzed by automated Edman degrada-tion, and radioactivity from each fraction was measured byscintillation counting.

RESULTS

Inhibition of �-Secretase Results in Accumulation of C-termi-nal Fragments of Pmel17—Previous studies have indicated thatthe release of the M� fragment from the lumenal domain ofPmel17 is a critical step in the formation of melanosomal stri-ations (3, 8). In this process, the remainingC-terminal fragmentof Pmel17 is apparently degraded rapidly (3, 8). In view of theloss of pigmentation in presenilin-deficient mice, we thereforeinvestigated whether presenilin-mediated �-secretase cleavagecould be involved in Pmel17 processing. We incubated humanMNT-1 as well as murine B16-F0 cells with increasing concen-trations of the �-secretase inhibitor DAPT. Immunoblottingwith a C-terminal antibody against Pmel17 (�Pep13h) demon-strated the expected P1 Pmel17 species at 100 kDa and the26-kDa M� fragment (3) (Fig. 1, B and C). Interestingly, weobserved a DAPT-dependent accumulation of a novel Pmel17

C-terminal fragment (Pmel17-CTF) in both cell lines (Fig. 1, Band E). Similar accumulation of Pmel17-CTF was seen with asecond �-secretase inhibitor LY450139 (data not shown). How-ever, this Pmel17-CTF cannot be detected at basal conditions,indicating that it is usually rapidly degraded. Time course stud-ies revealed that the levels of Pmel17-CTF started to increaseafter only 10 min of exposure to DAPT, again pointing to arapid turnover of this fragment under normal conditions (Fig.1F). Becausewe used prestainedmolecularmassmarkers in ourSDS-PAGE gels, the molecular masses are only approximate.Using unstained molecular mass markers, the detected molec-ular mass of�8–10 kDa suggests that Pmel17-CTF contains ata minimum the complete C terminus (5.2 kDa) and the trans-membrane domain (2.2 kDa) of Pmel17 (Fig. 1G). Because thisfragment specifically accumulated after �-secretase inhibition,it is likely, by analogy to APP and Notch processing (18, 19), torepresent a potential substrate for intramembrane proteolysis.The Pmel17-CTF is also considerably smaller than M�, whichimplies, again by analogy to Notch receptor and APP process-ing where there is an antecedent proteolysis by �-secretase orsimilar “sheddase” activity, that this peptide is generated after anew cleavage of Pmel17 C-terminal to the PC cleavage site. Wedesignate this cleavage as the S2 cleavage site.Because it has been shown that inhibition of �-secretase

results in a loss of pigmentation (16), we tested whether inhibi-tion of �-secretase has an impact on M� generation. Triton-soluble supernatants and the insoluble pellets extracted by SDSwere blotted with HMB45, an antibody that specifically recog-nizes sialylated Pmel17 present in P2 but not P1 species and inthe fibrillar matrix of melanosomes (2, 20, 21) formed by M�and its cleavage product M�C (8). Increasing concentrationsof DAPT resulted in a slight increase in the sialylated form ofPmel17, and to a minor extent, species at 40–50 kDa, thelatter representingmost likelyM�C fragments in the Triton-soluble fraction (Fig. 1C). We observed an identical patternusing the �-secretase inhibitor LY450139 (data not shown).However, the insoluble pellet did not show any significanteffects of DAPT treatment when immunoblotted withHMB45, indicating no overt role of �-secretase activity onM� generation (Fig. 1D).To further test the role of the �-secretase in the processing of

Pmel17, nicastrin expressionwas inhibited by siRNA treatmentinMNT-1 cells (17). As expected, therewas a strong decrease inthe levels of nicastrin and the PS1 N-terminal fragment, dem-onstrating that reduction in nicastrin expression led to reducedassembly of PS complex (17) (Fig. 2A). Consistent with theabove observations, there was marked accumulation ofPmel17-CTF after siRNA treatment without any changes in thelevels of Pmel17 P1 or M� species (Fig. 2A). Finally, we tran-siently expressed Pmel17 in mouse embryonic fibroblasts defi-cient in both PS1 and PS2 (MEF PS1/2�/�). The absence ofboth presenilin genes resulted in the accumulation of Pmel17-CTF at steady state basal conditions. But this alteration couldbe restored in MEF PS1/2�/� transfected with human PS1(Fig. 2B). Therefore, these results support the concept thatPmel17, or specifically Pmel17-CTF, is a substrate of�-secretase cleavage.

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Pmel17 Is a Substrate for �-Secretase-mediated Cleavage—Cleavage by �-secretase results in the release of an intracellulardomain (ICD) from type I membrane proteins (22). The half-life of these fragments, such as the APP intracellular domain,can be extremely short, most likely because of its rapid degra-dation by the proteasome or insulin-degrading enzyme (23, 24).To detect the release of Pmel17-CTF by �-secretase mediatedproteolysis, membrane fractions of MNT-1 cells were pre-treated with DAPT to first accumulate Pmel17-CTF and thenincubated at 37 °C without DAPT to allow the generation andrelease of the predicted Pmel17 intracellular domain fragment.Following immunoblotting with �Pep13h antibody, a new

6-kDa Pmel17 fragment wasdetected. This fragment was notseen when the membranes wereeither incubated at 4 °C or treatedwith DAPT (Fig. 2C). The additionof lactacystin, a specific inhibitor ofthe 26 S proteasome, had no effect,whereas EDTA increased the yieldof Pmel17-ICD slightly, most likelyby inhibiting residual activity of thecytosolic metalloprotease insulin-degrading enzyme (17). Theseresults therefore confirmed thatintramembrane cleavage of Pmel17-CTF is mediated by �-secretase,resulting in the release ofPmel17-ICD.S2 Cleavage Occurs Independ-

ently of PC Activity—It has beenshown that activity of a furin-likePC is required for formation ofintracellular Pmel17 fibrils (3). Totest whether the formation ofPmel17-CTF requires antecedentPC cleavage, we incubated MNT-1cells with the furin inhibitor Dec-RVKR-CMK for 48 h. After 24 h,DAPTwas added to inhibit Pmel17-CTF from further cleavage. Asexpected, generation of M� frag-ment was blocked after PC inhibi-tion, and there was an accumulationof P2 or the full-length, glycosylatedform of Pmel17, which is normallyrapidly proteolyzed by PC. At thesame time, the DAPT-dependentaccumulation of Pmel17-CTF wasunaffected (Fig. 3A). The sameresults were obtained by expressingPmel17-i and the recently describedPmel17-i PC site cleavage mutant(3) in HeLa cells (Fig. 3B). Thissuggests that cleavage at the S2site occurs independently of PCcleavage.S2 Cleavage of Pmel17 Is Induced

by Phorbol Ester—Because shedding of the extracellulardomain of transmembrane proteins is often stimulated byphorbol esters, we asked whether Pmel17 cleavage fragmentsare sensitive to phorbol esters andwhether this step is related toS2 cleavage of Pmel17. Incubation of MNT-1 cells with PMAresulted in decreased formation of M� (0.57 � 0.02 S.E.; p �0.05 by Student’s t test) (Fig. 3C). However, coincubation ofPMA and DAPT showed not only the decrease inM� fragment(0.54 � 0.01), just as seen in the control MNT-1 cells treatedwith dimethyl sulfoxide (DMSO), but also an increase in thelevel of Pmel17-CTF (2.19 � 0.25; p � 0.05). In addition, PMAincreased the secretion of the soluble Pmel17 (sPmel17) frag-

CC

FIGURE 1. Effect of the �-secretase inhibitor DAPT on Pmel17 processing. A, schematic diagram of Pmel17and epitopes of antibodies. Pmel17 contains five potential N-glycosylation sites indicated by branched struc-tures. The long form of Pmel17, Pmel17-l, is characterized by a seven amino acid insertion (VPGILLT) within thelumenal domain close to the transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a potentialN-glycosylation site near this insertion. The epitopes of antibodies �Pep13h and HMB45 are indicated. Cleav-age by a furin-like PC results in the formation of the M� and the membrane-bound 26-kDa M� fragment, whichare connected via disulfide bonds. Release and further processing of the M� fragment into M�N and M�Cfragments results in the formation of fibrils and marks the transition of stage I to stage II melanosomes (dashedline). B, human MNT-1 cells were incubated with increasing amounts of DAPT for 18 h, and then the lysates wereseparated by SDS-PAGE and analyzed by immunoblotting with �Pep13h antibody. DAPT treatment resulted inthe accumulation of a C-terminal fragment of Pmel17 (CTF), whereas Pmel17 P1 and M� fragment wereunchanged. C, probing the Triton-soluble fraction with HMB45 revealed increased amounts of the highlyglycosylated P2 form of Pmel17 after DAPT incubation. D, detection of Pmel17 amyloidogenic fragments (M�C)in the SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO cells treated with increasingconcentrations of DAPT. Immunoblotting using antibody �Pep13h revealed the formation of CTF of similar sizeas in MNT-1 cells. F, time course analysis of Pmel17, M�, and Pmel17-CTF after DAPT treatment. The cell lysateswere immunoblotted using �Pep13h. Pmel17-CTF was detectable after 10 min of incubation with 1 �M DAPT.G, the size of the Pmel17-CTF was determined using an unstained low molecular range peptide standard. Themarker peptides were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot using�Pep13h.

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ment into the medium (Fig. 3D). These results suggested thatPMA enhanced S2 cleavage, and consequently, the cleavageproducts, Pmel17-CTF and sPmel17, are correspondinglyelevated.Identification of the Pmel17-CTF Cleavage Site—To deter-

mine the position of the S2 cleavage site, we first testedwhetherthe C-terminal fragment is glycosylated because this wouldmake mass determination more difficult. By SDS-PAGE, weestimated the Pmel17-CTF to be �8–10 kDa. This size wouldencompass a potential N-glycosylation site (NVS) at position568 of Pmel17-i, 25 residues from the transmembrane surfacewithin a region where the S2 cleavage is predicted to take place(Fig. 4E). MNT-1 cell lysates treated with PNGase I digestionand immunoblotted with �Pep13h revealed that full-length

Pmel17 and M� fragment, but notPmel17-CTF, were reduced inmolecular masses after PNGase Idigestion. This suggested that eitherthe cleavage site is between theNVSsite at position 568 and the plasmamembrane or that this NVSmotif isnot glycosylated (Fig. 4A).We next performed mass spec-

trometry to determine the molecularmass of Pmel17-CTF. MALDI-TOFanalysis of �Pep13h immuno-precipitates from DAPT-treatedMNT-1 cells indicated a mass of8,840 Da for Pmel17-CTF (Fig. 4B).Alternative splicing results in thelong (Pmel17-l) and the more abun-dant short (Pmel17-i) isoforms thatdiffer by 7 amino acid residues (10)(Fig. 4C). Therefore, the presence ofboth isoforms may confound theanalyses of the cleaved fragments.To obtain definitive evidence of theS2 cleavage site, we metabolicallylabeled HEK293 cells transfectedwith Pmel17-i with [35S]methioninein the presence of DAPT. Immuno-blotting analysis showed compara-ble processing of Pmel17 inHEK293T and MNT-1 cells (datanot shown). After immunoprecipi-tation, the radiolabeled Pmel17-CTF was analyzed by radiosequenc-ing, which showed that the peak of35S radioactivity was recovered infraction 3 (Fig. 4D). Together withthe mass spectrometry results, weconclude that S2 cleavage ofPmel17-i occurs between Gln-583and Leu-584 (Fig. 4E), 12 residuesN-terminal to the predicted mem-brane surface, in agreement withknown shedding sites of other type Itransmembrane proteins (19, 25,

26). The resulting Pmel17-CTF has a theoretical molecularmass of 8,590 Da. The difference of �250 mass units betweenthe results from theMALDI analysis and the calculated molec-ular mass is likely due to unknown post-translational modifica-tions of the fragment.Generation of S2 Cleavage Site Mutants—The preceding

findings indicate that the S2 site is important for the amyloido-genic processing of Pmel17, likely by releasing the large intralu-minal fragment from the membrane where they are furtherproteolyzed and then assembled into the amyloid scaffold onwhichmelanin pigment is deposited. To investigate more thor-oughly the role of the predicted S2 site on amyloidogenic proc-essing of Pmel17, we generated and tested several Pmel17-iconstructs with mutations around this cleavage site. Specifi-

FIGURE 2. Pmel17 is substrate for �-secretase. A, MNT-1 cells were transfected with siRNA duplexes againstNicastrin on two consecutive days. 48 h after the last transfection, cell lysates were analyzed for nicastrin, PS1,tubulin, and Pmel17 by immunoblotting using antibodies N1660, 27G, E7, or �Pep13h, respectively. Transfec-tion with nicastrin siRNA duplex resulted in the accumulation of the Pmel17-CTF with no changes to P1 or M�fragment (right panel), together with a decrease in the levels of PS1-N TF and nicastrin (left panel). B, PS1�/�

PS2�/� mouse embryonic fibroblasts with or without stable transfection of human PS1 were transfected withPmel17-l-Myc construct. Absence of PS1 and PS2 resulted in the accumulation of Pmel17-CTF as detected byimmunoblotting with �Pep13h. C, crude membrane preparations of DAPT treated MNT-1 cells were incubatedfor 4 h at 4 °C with dimethyl sulfoxide (DMSO) or at 37 °C with dimethyl sulfoxide 1 �M DAPT, 1 mM EDTA or 1 �M

lactacystin. Pmel17-ICD production was monitored by immunoblotting with �Pep13h antibody. Incubation at37 °C resulted in the formation of a �6-kDa fragment that was absent at 4 °C or after treatment with the�-secretase inhibitor DAPT. Lactacystin and EDTA had no affect on Pmel17-ICD generation.

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cally, these mutations encode either amino acid substitutions(QL, 2D, and 4D) or deletions (Del4 andDel14) in Pmel17-i thatmight influence S2 cleavage (Fig. 5A). Cell lysates of Pmel1-imutants expressed inHeLa cellswere analyzed by immunoblot-ting. In the setting of DAPT treatment, we observed a markedreduction in the formation of Pmel17-CTF in all mutantsexpressed in HeLa cells (Fig. 5B). This suggests that perturbingthe S2 cleavage has substantial effects on decreasing the level of�-secretase substrate. This finding is not surprising, especiallyin light of the apparent requirement of an antecedent jux-tamembrane cleavage before intramembrane cleavage by�-secretase can occur (27). Moreover, all of the mutants exceptfor theQLconstruct showed significantly decreasedM� forma-tion, indicating that these mutations impacted PC cleavage.Importantly, all of the mutants showed a significant reductionin the levels of insoluble M�C fragments (Fig. 5C). Closerinspection of the QL mutant showed that whereas M� levels

were close to normal, there was asignificant 60% reduction in the lev-els of M�C fragments, suggesting aspecific impairment in the forma-tion of amyloidogenic M�C pep-tides when the predicted S2 site wasmutated to impair cleavage (Fig.5D). However, we did not observe adecrease in either the levels ofPmel17-CTForM�C fragments in aPmel17 construct containing thepreviously described PC site muta-tion (3) (Fig. 5, B and D). Further,introducing the same Q583L muta-tion into a construct where the PCsite was deleted resulted in a signif-icant 40% reduction in the levels ofM�C fragments (Fig. 5, D and E).Treatment of the QL/�PC mutantwith DAPT resulted in the samedecrease in Pmel17-CFT formationas observed in the QL mutationalone (Fig. 5F), again indicating thatthe effect of the QL mutation onPmel17-CTF generation is inde-pendent of PC cleavage.Modulation of S2 Cleavage

Changes M�C Levels—BecausePMA stimulation increased the for-mation of Pmel17-CTF, presumablyby enhancing S2 cleavage (Fig. 3C),we predicted that this effect shouldbe abolished in S2 cleavage sitemutants. Indeed, whereas coincu-bation of Pmel17-i-transfectedHeLa cells with DAPT and PMAincreased Pmel17-CTFs as seenbefore (Fig. 3C), the QL mutant didnot respond to PMA stimulation(Fig. 6A). Further, this mutationabolished the PMA stimulated

release of sPmel17 into the cell culture medium. Importantly,PMA also enhanced the generation of insoluble M�C frag-ments in Pmel17-I transfected cells, but this was absent in theQL mutant (Fig. 6A).To determine the class of protease that mediates S2 cleav-

age, we initially tried a number of protease inhibitors,including E64, 4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride leupeptin, aprotinin, EDTA, and pepstatin A,but were unable to detect any changes in the formation ofPmel17-CTF (data not shown). We then tested the morespecific hydroxamic acid-based metalloproteinase inhibitorTAPI-2, known to inhibit a variety of ADAM proteases.Treatment of HeLa cells transfected with Pmel17-i withTAPI-2 for 18 h led to 50% decrease in the level of Triton-insoluble M�C fragments. (Fig. 6C).We chose to investigate ADAM10 and 17 as candidate pro-

teases for their ability to mediate the formation of M�C frag-

FIGURE 3. PC and S2 cleavage occur independently. A, MNT-1 cells were incubated with the furin inhibitorDec-RVKR-CMK or dimethyl sulfoxide (DMSO) with or without DAPT. The cell lysates were analyzed by immu-noblot using antibody �Pep13h. Dec-RVKR-CMK inhibited the formation of M� but had no effect on the levelof Pmel17-CTF. Longer exposure (lower panel) showed the accumulation of the highly glycosylated P2 form ofPmel17 after Dec-RVKR-CMK treatment. B, HeLa cells were transfected with Pmel17-i or Pmel17-i constructcontaining a mutation at the PC cleavage site preventing the formation of M� (�PC). Treatment with DAPT didnot abolish Pmel17-CTF formation. C, MNT-1 cells were treated for 4 h with vehicle, DAPT, or Dec-RVKR-CMKwith or without 1 �M PMA. The cell lysates were immunoblotted using antibody �Pep13h. PMA stimulationdecreased the level of M� fragment slightly (0.54 � 0.01, n � 2) but increased the level of Pmel17-CTF (2.19 �0.25, n � 2). As seen above, PC inhibition resulted in the accumulation of highly glycosylated Pmel17 P2species. D, conditioned media from C (first and second lanes) were immunoblotted using HMB45. PMA stimu-lated the secretion of Pmel17.

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ments, because the vast majority of known ADAM substratesincludingAPP andNOTCHare processed by either one or bothof these ADAMs (28). siRNA targeting of ADAM10 andADAM17mRNAs led to a significant�43 and�63% reductionin the protein levels of ADAM10 and ADAM17, respectively(Fig. 6E; n � 3, p � 0.01 Student’s t test). Consistent with the

TAPI-2 result, siRNA knock-downof ADAM 10 or ADAM 17 bothresulted in �40% reduction in thelevels of M�C fragments normal-ized to P1 (Fig. 6D). Taken together,these findings indicate that phorbal-sensitive, mediated S2 cleavage bythe ADAM family of metallopro-tease is the critical step in releasingPmel17 from the membrane, afterwhich further proteolysis reducesM�C into fragments that assembleinto amyloid fibrils.

DISCUSSION

Pmel17 plays a central role inmelanogenesis by regulating thebiogenesis of the early stages of themelanosome. Release of the M�ectodomain leads to the formationof melanosomal striations (Pmel17amyloid) that has a promoting effecton melanin synthesis, presumablyby providing a scaffold for pigmentdeposition (11). Pmel17 fibers showtypical properties of amyloid withcross �-sheet structure, staining byamyloid-selective fluorophores, anddetergent insolubility. It has beenshown that recombinant M�ectodomain of Pmel17 assembles 4orders of magnitude faster thanother known amyloidogenic sub-strates like amyloid �-protein or�-synuclein, suggesting that therelease of this fragment has to behighly regulated to avoid cellulardamage (11). Proteolytic processingof Pmel17 by furin-like proproteinconvertase is required to generateM� and M� fragments, both ini-tially tethered to the vesicle mem-brane. It remains to be elucidatedhow the M� fragment is subse-quently released from the mem-brane and from the disulfide linkageto the membrane bound M� frag-ment. Nevertheless, without releasefrom the membrane tether, M�fragments cannot assemble intofibrils. In this study, we showed thata novel proteolytic activity cleaves

Pmel17 in a juxtamembrane position to release M� from themembrane anchor. This cleavage (between residues 583 and584), which we designate as the S2 site, is phorbol ester-sensi-tive and sheds the M� fragment and a portion of the M� frag-ment into the lumen. Following S2 cleavage, the now N-termi-nal truncated M� fragment (Pmel17-CTF) is quickly recleaved

FIGURE 4. Position of the S2 cleavage site. A, lysates of MNT-1 cells incubated with DAPT were digestedwith PNGase F or mock control. Immunoblotting with �Pep13h showed a decrease in the molecularmasses of P1 (P1*) and M� (M�*) but not Pmel17-CTF. B, cell lysates of MNT-1 cells treated with DAPT orvehicle (lower panel) were immunoprecipitated with �Pep13h antibody and analyzed by MALDI-TOF. Arepresentative MALDI-TOF tracing is shown. C, scheme of the juxtamembrane region of the alternativesplicing variants of Pmel17 expressed in MNT-1 cells. The methionines are underlined, and the transmem-brane domain is depicted in gray letters. D, Pmel17-i-Myc transfected HEK293 cells were metabolicallylabeled with 35S in the presence of DAPT. The cell lysates were immunoprecipitated using anti-Mycantibody. The precipitates were then separated by SDS-PAGE and transferred to a membrane, and thePmel17-CTF band was excised. Radioactivity was measured from the fractions after Edman degradation.The peak in radioactivity was found in fraction 3, suggesting a cleavage between Gln-583 and Leu-584 ofPmel17-I. E, scheme of the S2 cleavage site of Pmel17-i at Gln-583. The glycosylation side NVS is depictedin italic letters. The S2 cleavage (S2), the 35S-labeled Met-586 (35S), and the alternative splicing site areindicated. TM, transmembrane domain.

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by �-secretase, and the resultantPmel17-ICD fragment is then rap-idly degraded. Thus, our resultsshow that Pmel17 is a �-secretasesubstrate. However, �-secretaseactivity itself is not required for theformation of Pmel17 fibril forma-tion because it is the S2 cleavagepreceding �-secretase cleavage thatregulates the release ofM� and sub-sequent formation of Pmel17amyloid.Amyloid Formation of Pmel17

and S2 Cleavage—Because the oxi-dative environment within earlymelanosomes makes it unlikely thatthe disulfide bonds are reduced, ithas been suggested that that M�fragments are released from themembrane tether through proteo-lytic activity (4). Indeed, our resultsare entirely consistent with the lat-ter speculation. Specifically, weshowed that the lumenal domain ofPmel17 is liberated by a novel S2cleavage close to the plasma mem-brane. This cleavage results in therelease ofM� fragment and throughadditional proteolysis, the subse-quent generation of insoluble M�Cfragments. At the same time, anunstable 8.5-kDa C-terminal frag-ment derived from the residual M�fragment is generated (Fig. 7). ThisC-terminal fragment is unstablebecause it is quickly recleaved by�-secretase activity. The resultingPmel17-ICD peptide, like the other�-secretase released cytoplasmicdomains, is rapidly degraded.It has been reported that PC

cleavage of Pmel17 initiates theassembly ofM� and is presumably arequirement for the generation ofPmel17 amyloid fibrils (3). Never-theless, using the same PC sitemutant, we and others could notdetect reduced amounts of M�Cfragments (8). However, neitherHoashi and co-workers nor weexamined the cells by electronmicroscopy as was performed in theoriginal study, so these findingsmaynot be directly comparable. Fur-thermore, it was reported thatexpression of �1-antitrypsin Port-land (�1-PDX), an inhibitor of PCactivity, had a negative impact on

FIGURE 5. Mutation of the S2 cleavage site results in reduced Pmel17 insoluble aggregates. A, generationof Pmel17 mutants. Schematic diagram of Pmel17 S2 and PC site mutations. The transmembrane domain isdepicted in gray, amino acids substitutions are underlined, and deletions are shown as dashes. B, Pmel17-i-Mycmutants were expressed in HeLa cells in the presence of DAPT. The cell lysates were immunoblotted usinganti-Myc antibody. All of the S2 site mutations resulted in markedly reduced levels of Pmel17-CTF. C, M�Cfragments were reduced in all Pmel17 S2 mutants. HeLa cells were sequentially extracted with Triton X-100 andSDS. Equal amounts of protein were separated and immunoblotted with anti-Myc or HMB45 antibodies. All ofthe S2 site mutations resulted in reduced levels of M�C fragments but unchanged in the PC site mutant (�PC).D, Pmel17 QL mutant reduce M�C fragments independent of PC cleavage. The ability to form insoluble M�Cfragments was compared between wild type Pmel17, QL mutant (QL), PC site mutant (�PC), and a constructcontaining both mutations (�PC/QL). Expression of the PC site mutation had no impact on M�C fragmentsdetected with antibody HMB45, whereas the QL mutant reduced the formation by about 60%, even in thepresence of the PC site mutation within the same construct. Immunodetection of P2/M� in the soluble fractionshowed accumulation of P2 in the �PC mutants, which were was not observed in type Pmel17 or QL mutant(middle panel). Expression of full-length Pmel17 detected with antibody �Pep13h was comparable in all themutants as compared with control. In addition, M� fragment was absent in all �PC mutants as expected (upperpanel). E, quantification of experiment described in D. The graph shows the M�C to P1 ratio (n � 3 � S.E.,performed in duplicates; **, p � 0.01, Student’s t test). F, Pmel17 QL mutant reduced CTF levels independent ofPC cleavage. Constructs used in D above were expressed in the presence of DAPT and immunoblotted withanti-Myc antibody. The QL mutants lowered the levels of Pmel17-CTF, independent of PC cleavage.

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Pmel17 fiber formation (3). On the surface, these results areconsistent with the results obtained from the PC site mutant asreported in the same study (3). However, multiple groups haverecently demonstrated that several members of the ADAMprotease family need to be activated by proprotein converta-ses, such as furin (29–31), and that protein kinase C-depend-ent activation of �-secretase cleavage of APP also depends

on furin activity (32, 33). �-Secre-tase is a member of the ADAMprotease family, and its cleavage ofAPP is analogous to the S2 cleav-age site. Furthermore, the matrixmetalloprotease MMP-14, whichis PMA-inducible (reviewed inRef. 34), is cleaved by furin. Inter-estingly, MMP-14 is present in allstages of melanosomes (35) andhas been shown to be a sheddase(36). We therefore speculate thatexpression of �1-PDX preventsproprotein convertase-mediatedactivation of the S2 cleavageenzyme and, in so doing, inhibitsPmel17 fiber formation by pre-venting S2 cleavage.In this study, all of the mutations

at the S2 site showed significantreductions in the levels of M�Cfragments and Pmel17-CTF. It isunclear why these S2 mutants,except for the QL mutant, showedsuch a significant reduction in M�generation, especially when, in ourhands, deletion of the PC cleavagesite had no overt effect on M�C.Although we did not detect grosschanges in the processing of Pmel17biochemically ormorphologically inthese mutants (data not shown),there may be subtle changes inPmel17maturation that affected PCcleavage that we did not appreciate.Taken together, we hypothesize

that amyloid formation of Pmel17is dependent on S2 cleavage ofPmel17 and that the release of thisS2-cleaved ectodomain, whichincludes a portion of M� stilllinked to M� by disulfide bond, isthe initial precursor of Pmel17amyloid rather than M� itself.Because the observed size of M�Cfragments is smaller than thereleased 95-kDa ectodomain (37),further processing by carboxypep-tidase is necessary to truncate M�,a concept that is entirely consist-ent with our findings (8).

Characterization of S2 Cleavage Activity—Ectodomain shed-ding has been described for a variety of transmembrane pro-teins, mostly mediated by members of the ADAM family inparticular ADAM10 and 17 (28). In addition, the shedding ofa variety of substrates, like APP, TGF-�, Erb4, or Jagged, isstimulated by phorbol esters, presumably by activating pro-tein kinase C (38–42). It has been shown that a small frac-

FIGURE 6. Modulation of Pmel17 S2 cleavage by pharmacological treatment or knock-down of metallo-proteases. A, HeLa cells were transfected with Pmel17-i or the QL mutant. Incubation with DAPT or coincuba-tion with PMA for 4 h resulted in increased formation of Pmel17-CTF in case of the wild type construct but hadno effect on the S2 cleavage site mutant QL. Blotting of the conditioned media using HMB45 revealedincreased secretion of Pmel17 in cells transfected with Pmel17-i but not in the QL mutant. Likewise, PMAinduced the formation of insoluble M�C fragments in the insoluble fraction of Pmel17-i transfected cells butfailed to do so in the QL mutant. B, HeLa cells transfected with Pmel17-i were exposed to 100 �M of the inhibitorTAPI-2 for 18 h. The soluble and insoluble fractions were immunoblotted using anti-Myc or HMB45 antibodies,respectively. C, quantification of B (n � 2 � S.E., performed in duplicate; ***, p � 0.001, Student’s t test). D, HeLacells were cotransfected with Pmel17-i and control siRNAs or siRNAs targeting the metalloproteases ADAM10and ADAM17. Cell lysates were immunoblotted using Myc antibody for Pmel17. Pellet fractions were SDS-extracted and analyzed using antibody HMB45 (n � 3 � S.E., performed in duplicate; **, p � 0.01; ***, p � 0.001,one-way analysis of variance, Tukey post hoc test). E, the effectiveness of the siRNA knock-down was verified byimmunoblotting the lysates for expression of ADAM10 and ADAM17. Knock-down of ADAM10 resulted in adecrease of the proform (P) and the mature form (M). The latter was used for quantification (n � 3 � S.E.,performed in duplicate; **, p � 0.01, Student’s t test).

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tion of Pmel17 molecules is secreted into the extracellularspace as sPmel17 (37), although it is unclear whether thissecreted peptide has any biological function. Consistent withthe shedding of the extracellular domain of a number of typeI membrane proteins described above, sPmel17 shedding isalso phorbol ester-sensitive. Importantly, the phorbol estersensitive also extends to the release of M�C fragments (Fig.6A). It has been demonstrated that cAMP-elevating agentsand protein kinase C activation, such as the phorbol ester12-O-tetradecanoyl-phorbol-13-acetate, stimulate melano-genesis and increase the growth of cultured uveal melano-cytes (43). If this outcome is due to an increase in the releaseof Pmel17 from the membrane to generate more M�C frag-ments, then it is entirely consistent with our results showingthe stimulation of S2 cleavage and the coordinated increasedin insoluble M�C aggregates by PMA.

In addition, our results indicated that members of theADAM family of metalloprotease are likely candidates for thePmel17 S2 site enzyme, because treatment with TAPI-2 inhib-itor and siRNA targeting ofADAM10 andADAM17 all reducedthe level of insoluble M�C fragments. However, we cannotexclude that additional members of the large family of ADAMproteases are capable of performing this cleavage.Role of �-Secretase—Although we initially speculated that

�-secretase cleavage may be related to the release of the lume-nal Pmel17 fragment from the membrane, this is an unlikelyscenario in view of the topology of Pmel17. Because it is therelease of the N-terminal and not the C-terminal Pmel17 frag-ment that is critical formelanogenesis, �-secretase activity can-not mediateM� amyloid generation because the antecedent S2cleavage has to take place first and would have released M�

from themembrane. Given this, what is the role of presenilin inPmel17 metabolism? A signaling function of the intracellulardomain has been established for Notch (44), but it is still anopen question with most of the other �-secretase substrates(45). In this context, it has been proposed that �-secretase canfunction as the “proteasome of the membrane” by removingtransmembrane proteins destined for degradation (46). If so,the rapid turnover of the Pmel17-CTF certainly fits this modelof �-secretase function to facilitate the degradation of thesemembrane intermediates. Furthermore, the fast removal ofboth Pmel17-CTF and Pmel17-ICD after S2 cleavage is onereasonwhy these fragments have not been described before andalso explains the rapid disappearance of Pmel17 C-terminalimmunoreactivity as soon as the melanosomal striation isformed (3).Comparison with Other Amyloidogenic Substrates—There

are striking similarities and differences of this functional amy-loidogenesis to pathological amyloid formations. In the familialamyloidosis of Finnish type, mutated gelsolin requiressequential cleavage first by furin and then a second cleavageby a metalloproteases to release the mutant amyloidogenicpeptide (47). In wild type gelsolin, furin cleavage does notnormally take place (48), but only in the setting of the muta-tion. In the British form of dementia involving the BRI geneproduct, amyloidogenic processing also involves furin-me-diated cleavage to release the mutant BRI peptide (48). In thecase of Pmel17, PC cleavage participates in Pmel17 amyloidformation, but it appears that S2 cleavage is required to ini-tiate the membrane release. Other proteins may also beeninvolved in the amyloidogenic processing of Pmel17 (13),especially because S2 cleavage generates a fragment thatrequires further proteolytic activities to finally generateM�C. Further work will be needed to fully characterize theprotease activities responsible for S2 cleavage and to charac-terize how this cleavage is regulated physiologically, as wellas to determine the other proteases that truncate Pmel17into smaller M�C amyloid fragments. Studying the complexregulation of functional Pmel17 amyloid formationmay gen-erate new insights into pathological amyloids of misfoldingdiseases.

Acknowledgments—We are grateful to Dr. Michael S. Marks for pro-viding antibodies and cDNA constructs and to Dr. David E. Kang forproviding the presenilin-deficient fibroblast cells. We thank Dr. HuiZheng for helpful discussions and for sharing unpublished results. Wealso thankMatthewWilliamson (University of California, San DiegoProtein Sequencing Facility) for assisting in the radiosequencing andDr. Sarah Sagi and Barbara Cottrell for advice onmass spectrometry.The antibody E7 against tubulin developed by M. Klymkowsky wasobtained from the Developmental Studies Hybridoma Bank devel-oped under the auspices of the NICHD, National Institutes of Healthand maintained by The University of Iowa Department of BiologicalSciences.

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FIGURE 7. Proposed schematic processing of Pmel17. Pmel17 is firstcleaved by PC. The M� and M� fragments remain tethered by disulfidebonds. S2 cleavage then releases the Pmel17 ectodomain consisting ofM� and M�C fragments from the membrane into the lumen of melano-somes. The released ectodomain is then further cleaved into M�N andM�C fragments at presumably multiple cleavage sites (indicated by ques-tion marks). These proteolytic events result in smaller M�C fragments thatassemble into Pmel17 amyloid. After S2 cleavage, Pmel17-CTF undergoesintramembrane cleavage by �-secretase, and the released ICD fragment israpidly degraded.

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Amyloidogenic Processing of Pmel17

2306 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 4 • JANUARY 23, 2009

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Weggen and Edward H. KooMarkus P. Kummer, Hiroko Maruyama, Claudia Huelsmann, Sandra Baches, Sascha

-SecretaseγCleavage, and the Resulting C-terminal Fragment Is a Substrate for Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase

doi: 10.1074/jbc.M808904200 originally published online December 1, 20082009, 284:2296-2306.J. Biol. Chem. 

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