Proteomic Analysis of Early Melanosomes: Identification of Novel Melanosomal Proteins Venkatesha Basrur,†,⊥ Feng Yang,‡,⊥ Tsuneto Kushimoto,† Youichiro Higashimoto,† Ken-ichi Yasumoto,† Julio Valencia,§ Jacqueline Muller,| Wilfred D. Vieira,† Hidenori Watabe,† Jeffrey Shabanowitz,‡ Vincent J. Hearing,† Donald F. Hunt,X and Ettore Appella*,† Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852 and Pathology Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Received August 20, 2002
Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis. In this report, we used mass spectrometry and subcellular fractionation to identify protein components of early melanosomes. Using this approach, we have identified all 6 of the known melanosome-specific proteins, 56 proteins that are shared with other organelles, and confirmed the presence of 6 novel melanosomal proteins using western blotting and by immunohistochemistry. Keywords: melanoma • proteomics • melanosome
Introduction Melanin is a complex pigmented biopolymer that is synthesized in specialized membrane-bound organelles called melanosomes that are produced exclusively by melanocytes and by retinal pigment epithelial cells (reviewed in refs 1 and 2). The biogenesis of melanosomes and the regulation of melanin synthesis have been studied extensively by means of electron microscopy, biochemical, and immunohistochemical methods. On the basis of their morphology and degree of melanization, melanosomes have been classified into four developmental stages (I-IV). Various melanosome-specific proteins, such as tyrosinase, Trp1/Tyrp1, Trp2/DCT, OA1 and MART1, are delivered to stage I melanosomes following their processing through the endoplasmic reticulum (ER) and Golgi network, via several adapter protein complexes.3-5 Another melanosomespecific protein, gp100/Pmel17, which is a structural matrix protein, is delivered directly to the stage I melanosomes without being processed through the Golgi network.6 Even though * To whom correspondence should be addressed. Ettore Appella, M. D., Laboratory of Cell Biology, Building 37, Room 1B03, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA. Phone: (301) 402-4177. Fax: (301) 496-7220. Email:
[email protected]. † Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health. ‡ Department of Chemistry, University of Virginia. § Center for Biologics Evaluation and Research, Food and Drug Administration. | Pathology Section, National Heart, Lung and Blood Institute, National Institutes of Health. ⊥ V.B. and F.Y. contributed equally to this work. X Department of Chemistry and Pathology, University of Virginia. 10.1021/pr025562r CCC: $25.00
2003 American Chemical Society
active melanogenic enzymes are found in the sorting vesicles and can be detected in stage I melanosomes, melanin synthesis does not begin until after gp100 is processed from its membrane bound into its soluble form and is integrated into the fibrillar matrix characteristic of stage II melanosomes.4,7 Stage III and IV melanosomes are mature forms that are laden with increasing amounts of dark melanin pigment. As the pigment is synthesized and deposited on the melanosome fibrils, the melanosomes move from the perinuclear region toward the periphery of the melanocytes, i.e., the dendrite tips.8,9 This intracellular movement of melanosomes is mediated in part by microtubular motor myosin Va that is recruited by rab27a via its interaction with melanophilin, a process that is only now being clarified at the molecular level.10-12 Once they arrive at the dendritic tips, melanosomes are captured by actin filaments and are transferred to adjoining keratinocytes.9,13 Despite the cloning and characterization of several key structural and enzymatic proteins that make up these organelles, the mechanisms involved in the biogenesis of melanosomes, the regulation of melanin biosynthesis and their transfer to keratinocytes are still only partially understood. The presence of melanosomes uniquely in melanocytes does not reveal whether they are generated by a novel or an existing cellular mechanism for organelle biogenesis. The observation that many human diseases affect pigmentation and lysosomal function suggests that both types of organelles are closely related.14-16 However, Raposo et al. have recently reported that melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes, within pigmented cells.17 Journal of Proteome Research 2003, 2, 69-79
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research articles To date, the identification and cloning of melanosomal proteins has involved the genetic approach of characterizing genes involved with melanosome/lysosome-related organelle dysfunctions, such as Hermansky-Pudlak syndrome (HPS), Griscelli syndrome (GS), and Chediak-Higashi syndrome (CHS) or the use of different mouse coat color mutants. Over the past 40 years, only 6 proteins have been identified as melanosomespecific proteins, whereas more than 100 distinct gene products are thought to play direct or indirect roles in regulating the formation and function of these organelles in melanocytes. Hence, a more direct approach to comprehensively identify all of the proteins that constitute these organelles is likely to provide useful insights into the complex mechanisms governing mammalian pigmentation and melanosome biogenesis. With the increasing availability of larger genomic and proteomic databases, the mass spectrometric approach to protein identification is fast becoming the preferred technique. Because the tandem mass spectrometer is able to sequence complex mixtures of proteins with great sensitivity, it can be used to analyze the proteomes of complex organelles, such as melanosomes. Hitherto, melanosomes have resisted such a direct analysis, as it was difficult to prepare sufficient quantities of highly purified, relatively melanin-free melanosomes. Stage III and IV melanosomes, which can be readily purified because they sediment to the bottom of sucrose density gradients, are not suitable for such analysis because the endogenous melanin renders many of the melanosomal proteins insoluble by covalent modification.18,19 Previously, we reported the capability of free flow electrophoresis to purify early (stage I and II) melanosomes from mixtures partially purified by sucrose density gradient centrifugation.4 Those purified early melanosomes were characterized using electron microscopy, immunohistochemical, and biochemical methods which clearly demonstrated that the processing of membrane bound gp100 into a soluble form is a critical step in the transition from stage I to stage II melanosomes, and that this then stabilizes melanogenic enzyme function allowing the start of melanin synthesis.4 In this report, we have used mass spectrometry in conjunction with free-flow electrophoresis (FFE) of sucrose density gradient fractions to identify protein components of early melanosomes as they hold the key to understanding the biogenesis and sorting mechanisms involved. Using this approach, we have identified all 6 of the known melanosomal proteins and several novel proteins whose presence in melanosomes has been confirmed by western blotting and by immunohistochemistry.
Materials and Methods Cell Culture and Purification of Early Melanosomes from Human MNT1 Cells. The pigmented MNT1 cells were cultured at 37 °C in an atmosphere of 95% air, 5% CO2 in 150 cm2 culture dishes in minimal essential medium (all culture reagents were from Life Technologies, Grand Island, NY), as previously detailed.4 Confluent monolayers of MNT1 cells were harvested with 0.05% trypsin, 0.53 mM EDTA and were washed once in 0.25 M sucrose by centrifugation at 1000g for 5 min at 4 °C. They were then homogenized on ice using 20 strokes of a glass:glass tissue grinder and centrifuged at 1000g for 10 min at 4 °C. Tissue homogenates were then fractionated using various protocols, as detailed below and outlined in the Figures. Melanosomes at various stages of maturation were separated as described previously4 using sucrose density gradient cen70
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trifugation (Gradient 1). Briefly, the homogenate was layered on a discontinuous gradient of 1.0, 1.2, 1.4, 1.5, 1.6, 1.8, and 2.0 M sucrose in 10 mM HEPES buffer, pH 7.0. Centrifugation was carried out at 100 000 ×g in a Beckman SW28 swingingbucket rotor for 1 h at 4 °C and melanosomes banding at 1.01.2 M sucrose were carefully removed and were further purified by FFE. Early melanosomes recovered from the 1.0-1.2 M sucrose interface were injected into the right inlet of an Octopus-PZE FFE apparatus (Weber GmbH, Kirchheim, Germany) at 2.0 mL/hr. FFE was performed at 1000-1100 V and approximately 110-125 mA using 0.25 M sucrose in TEA, pH 7.4, as the chamber buffer and an elution flow rate of 3-4 mL/ min. The temperature of the sample was kept at 4 °C, and the chamber was maintained at 10 °C. Fractions were collected and analyzed as detailed below. Subsequently, a more effective separation method was developed. Briefly, the MNT1 homogenate was resuspended in 2 M sucrose and was layered at the bottom of the 1.0-2.0 M sucrose step gradient noted above (this reverse gradient is labeled Gradient 2). The gradient was centrifuged at 100 000 ×g for 1 h as noted above and the 1.0 M fraction was carefully recovered. That 1.0 M sample was then layered carefully in the middle of a 3 part gradient (Gradient 3) consisting of equal volumes of 0.8, 1.0, and 1.2 M sucrose. That gradient was again centrifuged at 100 000 ×g for 1 h and all 3 sucrose fractions were recovered and analyzed by electron microscopy and by Western blotting as detailed below. Protein Digestion and Mass Spectrometry. Enriched stage I and II melanosomes were processed for the purpose of sequencing in two ways (Figure 1). First, we used SDS-PAGE and in gel digestion followed by 1D LC-MS/MS. Briefly, melanosome preparations were solubilized directly in sample buffer and 15 µg were separated on 10% NuPAGE gels according to the manufacturer’s specifications. Gels were stained with colloidal Coomassie stain (Invitrogen, Carlsbad, CA) for 6 h and were destained in water. The lane containing the sample was cut into 4-mm slices, irrespective of the extent of staining, from top to bottom of the gel and destained for further 3 h in 30% methanol. In gel proteolysis with modified, sequencing grade trypsin (Promega, Madison, WI) was carried out essentially as described previously. Briefly, the gel slices were diced into 1 mm cubes and washed with 150 µL 50% acetonitrile in 0.1 M ammonium bicarbonate buffer, pH 8.0. The diced cubes were dried and re-swollen in a minimal volume of 0.1 M ammonium bicarbonate buffer containing 0.5 µg trypsin. Digestion was carried out for 16 h at 37 °C with an additional aliquot of 0.25 µg trypsin added after 12 h. Peptides were extracted with 150 µL 60% acetonitrile containing 0.1% TFA for 30 min, followed by a further extraction with acetonitrile. All extracts were pooled and concentrated to about a 10-µL volume in a vaccufuge. About 2 µL of the digest was separated on an Aquasil C18 reverse phase column (15 µm tip × 75 µm id × 5 cm Picofrit column, New Objectives, Inc) using an acetonitrile/1% acetic acid gradient system (5-75% acetonitrile over 25 min followed by 95% acetonitrile for 3 min) at a flow rate of 300 nl/min. Collision induced dissociation spectra for the most abundant peaks were collected using an ion-trap mass spectrometer (LCQ Classic, ThermoFinnigan). The mass spectrometer was set for analyzing the positive ions and was operated on double play mode in which the instrument was set up to automatically acquire a full scan and a tandem MS/MS spectrum (relative collision energy ∼30%) on the most intense ion from the full MS scan. Dynamic exclusion was set to collect two MS/MS
Proteomic Analysis of Early Melanosomes
Figure 1. Outline of melanosome purification. This flowchart shows the method used to isolate melanosomes at various stages of maturation from MNT1 melanoma cells; the protocol involves homogenization of the cells, purification over a sucrose density gradient (Gradient 1) by ultracentrifugation, and (for Stage I and II melanosomes) further purification by FFE.4 The purified samples were then analyzed by SDS-PAGE followed by tandem mass spectrometry, or by direct solubilization and 2DLC tandem mass spectrometry as indicated.
spectra on the most abundant peak and then exclude it for 2 min. An initial database search against a subset of indexed human database was carried out using TurboSEQUEST. The results of TurboSEQUEST were manually verified and any uninterpreted collision induced dissociation (CID) spectra were manually searched using the MS-Tag provision of Protein Prospector (http://prospector.ucsf.edu)21 against a subset of human/mouse proteins in the nonredundant or SwissProt database. In solution digestion of proteins and removal of melanin followed by 2D LC-MS/MS was performed in the following manner: Melanosome proteins from 4 × 108 cells were solubilized in 100 µL of ammonium bicarbonate buffer (100 mM, pH 8.5), vortexed for 2 min, sonicated for 30 min, and then pelleted by centrifugation at 14000g for 10 min. The supernatant was digested with modified trypsin (5 µg) (Promega) at 37 °C for 4 h and then at room temperature for an additional ∼18 h. The digest was then acidified and frozen at -35 °C until analyzed. The membrane-protein pellet was solubilized in 10% SDS (50 µL) and 10 M urea (50 µL), and delipidated with a methanol/chloroform mixture as described previously.22 The above solution was treated with methanol (260 µL), mixed briefly, treated with chloroform (87 µL) and stirred to yield a single phase. The addition of water (174 µL), with vigorous mixing, afforded two phases that were separated into two layers, with a precipitate at the interface, by centrifugation
research articles (10000g for 2 min). The bulk of the upper aqueous methanol phase was removed and the pellet was washed quickly with methanol (40 µL) to remove pigmented material and was then treated with 150 µL of methanol and 100 µL of water. The resulting suspension was mixed vigorously and then centrifuged (10000g for 1 min). The supernatant was removed and the protein pellet was dried under vacuum on a Speed Vac and redissolved in a mixture of 10 M urea (35 µL) and 10% SDS (25 µL), reduced with 40 mM dithiothreitol (10 µL) at 37 °C for 1 h, and carboxyamidomethylated with 400 mM iodoacetamide (12 µL) in the dark at room temperature for 1 h. For proteolysis, the sample was diluted with 100 mM ammonium bicarbonate (400 µL) and digested with endo-lys-C (2.6 µg)(Roche) at 37 °C for 15 h at pH 8.5. Following dilution of the sample with an additional aliquot of 100 mM ammonium bicarbonate (2 mL), the mixture was then incubated with trypsin (5 µg) at 37 °C for 12 h, acidified to pH 3 with glacial acetic acid and desalted using a Poros 20 R2 column (360 × 200 µm) (Perseptive Biosystems, Cambridge, MA). Peptides were eluted from the column with 50 µL acetonitrile (80%) in 0.1% acetic acid. The eluate was then evaporated to dryness on a Speed Vac and resuspended in 0.5% acetic acid (pH 3) before loading it onto a Poros 20 HS cation exchange column (360 × 200 µm). After washing the HS column with 100 µL acetonitrile (40%) in 1% acetic acid (pH 3) and then with 100 µL 0.1% acetic acid, peptides were eluted stepwise with 100 µL of 0 mM, 75 mM and 500 mM KCl in 5 mM potassium phosphate buffer (pH 3) containing 5% acetonitrile. For 2D LC-tandem MS, aliquots of the above digests were loaded onto nano-HPLC precolumns (360 × 100 µm fused silica packed with 5-cm C18 beads (YMC ODS-AQ; Waters) and washed with 0.1% acetic acid. Precolumns were then connected to analytical columns (360 × 50 µm fused silica packed with 7-cm C18 beads (YMC ODS-AQ; Waters),23 and the samples were analyzed on a ThermoFinnigan, LCQ Deca mass spectrometer equipped with a nano-HPLC microelectrospray ionization source as described previously.24 Antibodies. Polyclonal antibodies to VAT-1 (Cys-MQEKKNVGKVLLVPGPEKQN), oculospanin (Ac-MEEGERSPLLSQETAGQKPLSVHR-Cys) and FLJ20420 (Ac-MGGTTSTRRVTFEADENE-Cys) were raised in rabbits against the respective KLHconjugated specific peptide sequences shown in parentheses. Sera from immunized rabbits were affinity purified using each appropriate peptide coupled with SulfoLink (Pierce Chemical Co, Rockford, IL). Specificities of antibodies were confirmed by ELISA and by immunoblot assays. Other antibodies used in this study included: RPEP7h for tyrosinase;25 HMB45 for gp100;26 RPEP13h for gp100;6 PDP for syntenin (a kind gift from Dr. Rouleau, Center for Research in Neuroscience, McGill University);27 and R-flotillin-1 (BD Biosciences, San Diego, CA). Western Blotting. Proteins (5 µg protein/lane) were separated on 8% or 14% SDS gels and were transferred electrophoretically to poly(vinylidene difluoride) membranes (Immobilon-P, Millipore Corp., Bedford, MA). The blots were incubated with primary antibodies (at 1/500 or 1/1000 dilution) for 1 h at 23 °C, washed several times, and subsequent visualization of antibody binding was carried out with Enhanced ChemiLuminescence (Amersham Corp., Arlington Heights, IL), according to the manufacturer’s instructions. Electron Microscopy. Specimens were fixed overnight at 4 °C in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. The samples were then Journal of Proteome Research • Vol. 2, No. 1, 2003 71
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Figure 2. Mass spectrometry analysis of melanosomal proteins. (A) Melanosomal proteins were resolved by SDS-PAGE, and the gel was sliced into 9 pieces as indicated by the brackets. The gel slices were digested with trypsin, and the peptides were analyzed by tandem mass spectrometry; the identified proteins are indicated. B) Total ion chromatogram (TIC; m/z 400-1500) obtained in the LC-ESI-MS analysis of one fraction (75 mM KCl) from the off-line cation exchange fractionation of trypsin-digested melanosomal proteins. Insets: ms/ms spectra of peptides from; (A) hypothetical protein FLJ20420 (Q9NX63), (B) melanocyte-specific protein (Pmel17 precursor, P40967).
stored in PBS at 4 °C until they were embedded in epoxy resin. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined using a Zeiss EM 912 electron microscope. For immuno-electron microscopy, fractions were reacted for 1 h at 23 °C with the primary antibody (at 1:100 dilution), 72
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washed twice in PBS, then incubated with the appropriate 10 nm gold-labeled goat anti-rabbit or anti-mouse IgG (at 1:25 dilution) for 1 h at 4 °C. Controls included samples with no primary antibody. The fractions were washed again twice in PBS, then fixed and processed for electron microscopy as described above.
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Proteomic Analysis of Early Melanosomes Table 1. Proteins Identified in Early Melanosomes
protein
identifi- theore- theoresequence accession cation tical Mr tical peptides coverage no. methoda (kDa) pI sequenced (%)
1 tyrosinase
P14679
1,2
60.4
5.7
3
10
2 TRP1/Tyrp1
P17643
1,2
60.8
5.6
13
35
3 TRP2/Dct
P40126
2
59.2
6.7
4
9
4 Pmel17/gp100
P40967
1,2
70.2
5.4
7
12
5 MART-1
Q16655
2
13.2
8.3
1
10
6 ocular albinism type 1 protein 7 Rab-7
P51810
1
43.9
7.5
4
12
P51149
1,2
23.5
6.4
4
23
8 Rab-27a
P51159
1,2
24.8
5.2
3
16
9 Rab-27b 10 Rab-38
O00194 P57729
1,2 1,2
24.6 23.7
5.4 7.6
1 4
5 18
11 LAMP-3/CD63 12 transmembrane glycoprotein HGFIN 13 VAT1
P08962 Q14956
1,2 1,2
25.6 62.6
8.1 6.2
3 3
8 7
Q99536
1,2
32.5
6.5
11
39
14 oculospanin 15 hypothetical protein FLJ20420 16 MDA-9/syntenin
Q9H1Z9 Q9NX63
1,2 2
36.5 26.1
5.6 8.5
6 1
22 6
O00560
1,2
32.4
7.1
6
34
17 flotillin 1 18 stomatin
Q969J8 P27105
1,2 1,2
47.4 31.7
7.1 7.7
5 7
13 30
19 flotillin 2 20 phosphatidylinositol 4-kinase type II
Q14254 14744398
1,2 1,2
41.7 54.1
5.2 8.5
2 4
5 9
21 22 23 24 25 26
P13473 P11279 P07339 P07858 O14773 Q92820
1,2 1,2 1,2 1,2 1,2 1,2
44.9 44.7 44.5 37.8 61.2 35.9
5.4 9.2 6.1 5.9 6.0 6.7
6 4 9 6 5 4
11 7 25 21 14 15
P04062 P38571 P07686 Q13510 1360694 Q9HAQ7
1,2 1,2 1,2 1,2 1 1
59.6 45.4 63.1 44.6 58.5 99.7
7.0 6.4 6.3 7.5 5.0 9.2
3 4 7 7 2 2
6 10 17 18 4 3
P09543
1,2
47.6
9.2
5
12
P38606
1,2
68.2
5.3
7
16
V-ATPase, subunit B V-ATPase, subunit D V-ATPase, subunit E V-ATPase, subunit S1 clathrin-coated vesicle/synaptic vesicle proton pump 116 kda subunit 40 reticulocalbin 1
P21281 P12953 P36543 Q15904 Q93050
1,2 1,2 2 1 1,2
56.5 40.3 26.1 52.0 95.8
5.6 4.9 7.7 5.7 6.2
5 4 3 2 7
12 14 16 3 12
Q15293
1,2
38.9
4.9
4
13
41 calumenin 42 ribophorin I 43 GRP 75/mortalin
O43852 P04843 P38646
1,2 1,2 1,2
37.1 68.6 73.8
4.5 6.0 6.0
8 3 3
26 5 6
44 45 46 47 48 49
GRP 78/BiP ERP99 GRP94 ERP28 PDI/THBP ERP57
P11021 P08113 P14625 P30040 P07237 P30101
1,2 1,2 1,2 1,2 1 1,2
72.3 92.5 92.4 28.9 57.1 56.8
5.1 4.7 4.8 6.8 4.8 6.0
9 7 4 4 5 12
17 11 5 18 10 27
50 51 52 53
ERP72 PDI A6 calreticulin calnexin
P13667 Q15084 P27797 P27824
1 1 1,2 1,2
72.9 48.1 48.1 67.5
5.0 4.9 4.3 4.5
2 2 8 6
4 5 24 12
P21796
1,2
30.7
8.6
6
24
27 28 29 30 31 32 33 34
LAMP2 LAMP1 cathepsin D cathepsin B tripeptidyl-peptidase I gamma-Glu-X carboxypeptidase glucosylceramidase acid lipase hexosaminidase B acid ceramidase saposin precursor ATP binding cassette, half transporter 2′,3′-cyclic nucleotide 3′-phosphodiesterase V-ATPase, subunit A
35 36 37 38 39
54 VDAC 1
localization/comments
melanosomal enzyme involved in melanin biosynthesis; type I membrane protein, mutations result in OCA1 melanosomal enzyme involved in melanin biosynthesis; type I membrane protein, mutations result in OCA3 melanosomal enzyme involved in melanin biosynthesis; type I membrane protein melanosomal protein; fibrillar matrix characteristic of melanosomes; type I membrane protein melanosomal protein; function unknown; type I membrane protein melanosomal protein; homology with G-protein coupled receptors; mutations result in OA13 LE/melanosome associated; suggested to be involved in Tyrp1 transport from LE to melanosome45 Rab27a involved in interacting with myosin Va through melanophilin;48 mutations result in Griscelli Syndrome. Distribution of Rab27b is similar to Rab27a on melanosomes;60 may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes61 melanosome; suggested to be involved in sorting of Tyrp1; mutation results in chocolate phenotype46 melanosome/lysosome; tetraspanin family could be melanogenic enzyme by similarity; belongs to pmel17/ NMB family62 abundant protein in synaptic vesicles; function unknown; shows ATPase activity and weak calcium binding property38,39,63 tetraspanin; unknown function40 unknown function syntenin is an adapter protein that couples transmembrane proteoglycans, syndecans, to cytoskeletal components enriched in detergent resistant lipid rafts; flotillin 2 is associated with filopodia formation64 lysosome/endosome, associated with noncaveolar rafts, probably required for AP2 containing clathrin coat assembly, specifically interacts with tetraspanin proteins65,66 lysosome; type I membrane proteins lysosome; aspartyl protease lysosome; thiol protease lysosome; belongs to peptidase family S53 lysosome; belongs to peptidase family C26 lysosome; Gaucher disease lysosome; Wolman disease lysosome; Sandhoff disease lysosome/endosome; Farber’s disease lysosome/microsome probably lysosome; ABC2 and ABCB9, a half transporter, have been shown to be present on lysosomes67,68
vacuole; hetero-multimeric enzyme complex responsible for acidification of various vacuolar compartments
secretory vesicles; Members of CREC family, EF hand proteins, localized to secretory pathway69 ER; type I membrane protein ER/mitochondria/cytosolic; involved in cellular senescence, glucose regulation, mitochondrial transport ER; chaperone ER; chaperones processing and transport of secreted proteins
ER; rearrangement of both interchain and intrachain disulfide bonds; ERP57 interacts with monoglycosylated glycoproteins in a disulfide-independent manner, probably via calnexin ER; chaperone ER; type I membrane protein; p90 calnexin similar to ER calnexin is found in melanosomes70,71 mitochondria/endosome/PM; Anion selective channel
Journal of Proteome Research • Vol. 2, No. 1, 2003 73
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Table 1 (Continued)
protein
glucosidase II R subunit Hsp60 CD59/protectin ACTA2 protein/actin, cardiac 59 Ras-related C3 botulinum toxin substrate 1/p21-Rac1 60 type II membrane protein 61 ubiquitin 55 56 57 58
62 microtubule-associated proteins 1A/1B light chain 3 63 galectin 3 64 Niemann-Pick C1 protein 65 homologue to hypothetical 19.5 kDa protein
identifi- theore- theoresequence accession cation tical Mr tical peptides coverage no. methoda (kDa) pI sequenced (%)
Q9P0 X0 P10809 P13987 Q13707
1,2 1,2 2 1,2
109.4 61.1 14.1 36.8
5.8 5.7 6.0 5.2
6 5 2 7
7 10 19 24
ER mitochondria; chaperone PM/vesicles
2
21.4
8.8
4
30
Q9Y2B0
1,2
20.7
4.8
2
16
P02248
1,2
8.5
6.6
3
44
1
14.5
9.0
1
6
PM; its homologue cdc42 is suggested to be involved in filopodia formation to facilitate the transfer of melanosomes to keratinocytes13 probably ER/lysosome; contains a signal peptide (1-21), saposin B motif (64-171) and C-terminal HDEL sequence apart from signaling proteins for proteosome mediated degradation, ubiquitin has also been shown to be involved in protein trafficking within the cell72 predominantly expressed in neurons and suggested to regulate the microtubule binding activity of MAP1A/1B
1,2 1,2
26.1 142.1
8.6 5.2
5 4
22 3
cytosolic/nuclear
1
31.2
8.8
2
10
human fragments: XP•087115: doesn’t have either of the peptides sequenced. But completely identical to the mouse protein from the middle to the end of the protein T34524, CAB59180: Have the second peptide sequenced
P15154
Q9GZQ8 Q96J47 O15118 12847703
66 nicastrin 67 LRP 130
Q92542 P42704
1,2 1,2
78.4 145.2
5.7 5.5
4 2
7 2
68 R-2-macroglobulin
P01023
1,2
163.2
6.0
3
2
a
believed to interact with proteins involved in cytoskeletal rearrangement and vesicle trafficking found at ∼30 kDa range
1 ) SDS-PAGE followed by in gel digestion and 1D LC-MS/MS; 2 ) In solution digestion followed by 2D LC-MS/MS.
Confocal Microscopy and Immunofluorescence. Dual labeling using immunofluorescence methods and laser scanning confocal microscopy was employed to evaluate the subcellular localization of proteins identified in this study, at the following dilutions: VAT-1 (1:20), oculospanin (1:25), FLJ20420 (1:25), HMB-45 (1:500), RPEP7 (1:500), RPEP13 (1:500), and flotillin-1 (1:10). Chamber slides containing subconfluent MNT-1 melanoma cells, were stained by the double indirect immunofluorescence method, as previously described in detail.4 Briefly, MNT-1 cells were washed twice with 1X PBS, and were then fixed with 4% paraformaldehyde for 15 min at 4 °C. All procedures were performed at room temperature (23-25 °C), except where indicated. After fixation, cells were washed twice with 1X PBS and were immediately permeabilized with 0.01% Triton ×100 for 3 min. Nonspecific binding was blocked with 5% normal serum for 1 h. Chamber slides were incubated with a mixture containing a polyclonal and a monoclonal antibody overnight at 4 °C. The polyclonal antibodies were reacted with secondary antibodies (goat anti-rabbit IgG; Vector; dilution 1:100) labeled with Texas red. The monoclonal antibodies were reacted with secondary antibodies (goat anti-mouse IgG; Vector; dilution 1:100) labeled with fluorescein, followed by nuclear counterstaining with DAPI (Vector Labs, Burlingame, CA). Reactivity was classified into three categories, according to whether they showed green, red, or yellow fluorescence. The latter was indicative of colocalization of the red and green fluorescent signals. All preparations were examined with a confocal microscope (model TCS-SP/DMRE; Leica, Heidelberg, Germany), equipped with HeNe, Argon and krypton laser sources.
Results Purification of Early Melanosomes and Protein Identification by Mass Spectrometry. Figure 1 shows a schematic summarizing the approach used to purify and characterize melanosomal proteins. Basically, the procedure takes advantage of the density of small amounts of melanin in early melano74
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somes to purify those organelles over stepwise density gradients (from 1.0 to 2.0 M sucrose). Earlier, we reported the use of FFE to further separate the 1.0 M sucrose fraction (which contains Stage I and Stage II early melanosomes) into tyrosinase-rich and protein-rich fractions.4 The specific activity of tyrosinase was enriched about 80-fold in the early melanosomes and the fraction was negative for mitochondria, Golgi, and early endosome markers, as determined by Western blot analysis. Ultrastructural analysis further confirmed that the fraction was highly enriched for stage II melanosomes. This FFE-purified early melanosome fraction was used for protein identification by mass spectrometry. Our early repeated attempts to establish a reproducible profile of melanosomal proteins using 2D PAGE were unsuccessful due to the presence of small amounts of the charged, insoluble polymer, melanin. The pigment, by virtue of its ability to covalently modify proteins,18,19,28,29 renders them insoluble and because many of the melanosomal proteins are membrane bound, under the mild solubilization conditions required for the IEF step of 2D electrophoresis, they are not efficiently solubilized. Hence, two different approaches were undertaken to comprehensively identify the proteome of the melanosome. In the first approach, FFE-enriched melanosomes were separated by SDS-PAGE, followed by in gel digestion and tandem mass spectrometry. As shown in Figure 2A, the melanosomal extract was resolved by SDS-PAGE into a relatively large number of protein bands, although many of those were poorly resolved from each other. Thus, we cut the gel into 9 slices and analyzed those slices by the in gel digestion technique which gave positive identification of 61 proteins (this experiment was repeated twice with comparable results). The protein characteristics and the peptides sequenced for each protein using this approach are listed in Table 1 (method 1). However, due to limited resolution of proteins due to the minor amounts of melanin and the presence in the digests of many other protein bands in low abundance, we resorted to a second
Proteomic Analysis of Early Melanosomes
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Figure 3. Further purification and analysis of melanosomal proteins. (A) Schematic of the preparation procedure utilized for further purification of Stage I and Stage II melanosomes; homogenates of MNT1 cells were suspended in 2 M sucrose and layered at the bottom of the discontinuous gradient (Gradient 2). Following centrifugation, the 1.0 M sucrose fraction, which contained stage I and II melanosomes, was recovered and layered in the middle of an extended gradient containing only 0.8, 1.0 and 1.2 M sucrose (Gradient 3). Melanosomes sedimenting in the 1.0 M sucrose fraction were recovered and then analyzed further by electron microscopy and Western blotting. (B) Ultrastructural analysis of the 1.0 M sucrose fraction from Gradient 3; sample contains only recognizable Stage II melanosomes. (C) Western blotting analysis of the original MNT1 homogenate, various fractions from Gradients 1 and 3, and from FFE; antibodies used to probe the fractions were specific for melanosomes (tyrosinase), mitochondria (MTC02) or endoplasmic reticulum (Bip).
approach, which is based on solubilization of proteins after melanin removal. Melanosomes were successfully solubilized as detailed in the Methods section and 2D-LC-tandem MS was performed. An example chromatogram depicting the identification of gp100 and FLJ20410 using this approach is shown in Figure 2B. Table 1 shows the complete repertoire of melanosomal proteins identified using these approaches. Sequencing was performed on at least 3 different melanosome preparations and only those proteins which were consistently observed either by 1D-LC and/or by 2D-LC approaches are reported in Table 1. Using both techniques, we have now identified a total of 68 proteins in melanosomes which include all 6 of the known melanosomespecific proteins (tyrosinase, Tyrp1, Dct, OA1, gp100, and MART1) whose presence (except for OA1) had already been confirmed by Western blot analysis.4 It is evident from these results that in addition to the known melanosomal proteins, there are a number of other proteins present in melanosomes, most of which are membrane-bound or membrane-associated proteins. These include several type I membrane proteins (LAMP-1 and LAMP-2), tetraspanins (CD63 and oculospanin), lipid raft associated proteins (stomatin, flotillin-1 and flotillin-2), vacuolar proton ATPases (VATPase A, D, B, E, and S1 and the clathrin coated vesicle/synaptic vesicle proton pump), and cytoskeletal and associated proteins involved in vesicular trafficking (actin, MAP1B LC3 and LRP130,
small GTPase family members or related proteins such as rab7, rab27b, rab38, and p21-rac1). Interestingly, with the exception of calnexin,30 which was identified by antibodies raised against purified melanosomes, no other ER proteins have been previously reported to be present in melanosomes. To confirm that ER proteins are indeed associated with melanosomes and do not represent contamination with other organelles, we devised a two-part reverse sucrose density gradient to further purify the melanosomes (Gradients 2 and 3 in Figure 3A). The reversed gradient 2 (which purifies melanosomes loaded in 2 M sucrose at the bottom of the gradient) greatly improved the purification of early melanosomes, completely removing mitochondria and other organelles. Final purification was achieved by recovering the early melanosomes from the 1.0 M sucrose fraction and placing them in the middle of an extended gradient (Gradient 3). Ultrastructural analysis (Figure 3B) of the 1.0 M sucrose fraction recovered from Gradient 3 clearly indicated that it consists of highly purified Stage II melanosomes, which are quite distinctive due to their internal fibrillar matrix. Western blot analysis (Figure 3C) shows that this early melanosome fraction as well as the 1.8 M sucrose melanosome fraction, which has been regarded traditionally as pure heavily melanized (stage IV) melanosomes, were positive for ER marker proteins, indicating that those ER proteins do not represent contamination, but are actually associated with melanosomes. Journal of Proteome Research • Vol. 2, No. 1, 2003 75
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To confirm the subcellular localization of these proteins in melanosomes, we used RPEP13 and HMB45, antibodies that recognize GP100 in Stage I or in Stage II melanosomes, respectively,4 with confocal immunohistochemistry. Antibodies to tyrosinase, Tyrp1 and Dct, which are traditionally used as specific markers of melanosomes, and which recognize these proteins when they are in transit in the ER and other vesicular sorting structures, were also included in our analysis (Figure 5). Dual labeling showed that tyrosinase, TYRP1, VAT-1, oculospanin, flotillin, and FLJ20420 co-localized (cf yellow color) with Stage I melanosomes (left column) and/or Stage II melanosomes (middle and right columns). The punctate distribution and extensive co-localization of VAT-1, oculospanin, flotillin, and FLJ20420 with the melanosomal markers indicates their association with early melanosomes. Note that the ER markers Bip and KDEL are not only found in the perinuclear area and co-localized with gp100 in early melanosomes, but are also readily detectable in mature melanosomes in the extremities of the melanocytes.
Discussion
Figure 4. Novel protein analysis in melanosome fractions. Fractions as detailed for Figure 3 were analyzed by Western blotting using antibodies specific for tyrosinase, VAT-1, oculospanin, syntenin, FLJ20420, and flotillin, as noted in the Figure.
Although mitochondria tend to co-purify with early melanosomes in Gradient 1, they were completely removed from early melanosomes purified through Gradients 2 and 3, as assessed by Western blot analysis and by electron microscopy. In addition, the 1.0 M fraction from Gradient 3 was highly enriched for the melanosome and ER markers (tyrosinase and BiP, respectively), but was completely negative for the mitochondrial marker (MTC02). Subcellular Localization of Novel Melanosomal Proteins. Because it is not practically feasible to validate all proteins identified in our preparation (Table 1) as melanosome specific or associated, we focused on a few of them. To determine if they are bona fide melanosomal proteins and to determine if they co-purify with tyrosinase, the most stringent marker of melanosomes, we used Western blotting with specific antibodies to those proteins (Figure 4). Fractions examined included whole cell lysates, the 1.8, 1.4, 1.2, and 1.0 M sucrose fractions from Gradient 1, the 1.0 and 0.8 M sucrose fractions from Gradient 3, and the purified FFE fractions (the tyrosinase-rich Fraction II and the protein-rich Fraction I). Western blot analysis clearly showed co-purification of VAT-1, oculospanin, syntenin and flotillin with tyrosinase-rich melanosomes. The apparent decreases in the intensities of reactive bands in the 1.8 M fraction (containing Stage IV melanized melanosomes), especially with respect to tyrosinase, oculospanin, and syntenin, is probably due to epitope masking by melanin deposition, as we have previously discussed.4 FLJ20420 and flotillin were not appreciably enriched in the tyrosinase containing fractions, which suggests that while they are associated with melanosomes, they may not be melanosome-specific proteins, but might just be generally present in many membrane-bound organelles. 76
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This study has demonstrated the utility of combining FFE with sensitive mass spectrometry techniques to analyze complex subcellular components, such as the melanosome, which shed light on its biogenesis. The combination of the two approaches for protein identification, such as in gel or in solution digestion followed by 1D-LC or 2D-LC MS, respectively, has resulted in the reproducible identification of proteins varying over a wide dynamic range in abundance (from the most abundant Tyrp1 to the low abundant MART1). Table 1 details the number of peptides which have been identified for each protein and whether they were identified by both techniques. In addition, the coupling of subcellular localization using Western blot analysis and confocal immunohistochemistry allows the further characterization of novel proteins not only in melanosomes, but in any organelle of interest. Ideally, one would like to have a suitable control to use for comparison but since the melanosome is such a unique organelle, there really is not such a control. To get around this problem we have used Western blotting and immunohistochemistry to show that the six novel proteins identified in this study do in fact colocalize with melanosomes. Proteomic mapping of early melanosomes has identified all six of the known melanosomal proteins as well as a number of other common constituents from lysosomes, endosomes and ER. This agrees with a number of earlier studies suggesting that early Stage I melanosomes are derived directly from the ERGolgi/endosome network and/or from lysosomes. A link between lysosomes and melanosomes has been widely accepted in the field and hence finding lysosomal and ER proteins was not surprising. Interestingly, a number of novel protein components were also identified, as summarized in Figure 6. Hence, our approach opens the way for the first time to address their functions in the melanosome and allows new proteins identified (such as VAT-1, oculospanin, syntenin, FLJ20420, and flotillin) to be analyzed for their roles in melanosome structure and/or function. Our results have already provided some important insights into the cellular machinery used to generate this specific organelle, which shows an admirable conservation of cellular processes and energy. At this stage of the analysis, it has become clear that the majority of components of melanosomes
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Figure 5. Subcellular localization of novel proteins. The subcellular localization of novel proteins was assessed by confocal immunohistochemistry. The marker used for Stage I melanosomes was RPEP13 (red, left column), whereas HMB45 was used as a marker for Stage II melanosomes (green, middle and right columns). Antibodies for melanosomes (tyrosinase and Tyrp1) and for ER (KDEL and Bip) are shown, as is the localization of novel proteins (VAT-1, oculospanin, syntenin, FLJ20402, and flotillin). Yellow indicates colocalization of both antibodies. Shown are the merged images of the red (Texas Red) and green (FITC) antibody stains; yellow indicated colocalization of both antibodies noted.
derive from both lysosomal and ER vesicles, are incorporated into early melanosomes, and that only a limited number of additional specific proteins are to be found that provide the melanosome with its unique architecture and function. Our findings, therefore, are consistent with the ultrastructural morphology of melanosomes which begin life as small amorphous spherical organelles often seen blebbing from the ER.31-34 These Stage I melanosomes have been shown to be positive for GP100 and ER markers.4 As other melanosome-specific proteins (such as tyrosinase, TYRP1, and DCT) are delivered via sorting vesicles to melanosomes, these vesicles coalesce with the maturing melanosomes and the size of that organelle gradually increases. Once the organelle matures to Stage II and becomes competent to produce melanin, the size of the melanosome remains relatively constant and further growth by vesicle fusion is minimal.35-37 The functions of the known melanosome-specific proteins are relatively well-characterized as structural or enzymatic components that play roles in the synthesis and deposition of melanin. In that context, it is interesting to speculate about
the putative functions of other proteins identified in this analysis as to what might be their function in melanosomes. For example, VAT-1 was identified more than a decade ago as an abundant membrane protein from Torpedo cholinergic synaptic vesicles; since then, it has been reported that VAT-1 exhibits ATPase38 and low affinity calcium binding activity.39 Whether VAT-1 plays a role in melanocytes with respect to the exocytosis of melanosomes is an exciting possibility to examine in future studies. As another example, oculospanin belongs to the tetraspanin superfamily, which includes 25 other members.40 Interestingly, oculospanin has a sorting signal sequence at its amino terminus (GERSPLL) that closely resembles the sorting signal sequence found in the carboxyl terminus of GP100 (GENSPLL) responsible for its targeting to the melanosome. Because one of the striking features of the tetraspanins is their ability to form multi-molecular complexes with each other and with other surface proteins which has led to the concept of a “tetraspanin web”, 40 oculospanin along with CD63 may be involved in the Journal of Proteome Research • Vol. 2, No. 1, 2003 77
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Figure 6. Schematic of melanosomal proteins identified in this study. Hypothetical figure depicting melanosomal proteins identified in this study (as listed in Table 1), grouped according to their anticipated function (e.g., pH regulation) or their presence in other organelles. The functions of some proteins shown are well-defined, others are conjectural based on predicted properties.
recruitment of organelle specific proteins and/or membrane remodeling which is crucial for melanosome movement and transfer. Further, a number of small GTPases were identified in our study. Many Rab GTPases have only very recently been shown to play important roles in melanosome protein sorting and movement within melanocytes.41-44 For example, rab7 and rab38 have been implicated in the intracellular sorting of Tyrp1,45,46 which is distinct from the sorting of tyrosinase and Dct to that organelle. The localization pattern of rab27b, a member of the rab27 subfamily, which consists of rab27a and rab27b, closely resembles that of rab27a in melanocytes42,47,48 which suggests that the two rab27 proteins are functional homologues. Recently, it was demonstrated that rab27a, which localizes on the melanosome membrane, acts as a receptor for myosin Va through its interaction with melanophilin.10,11,47-49 We do not know whether the a and b forms of rab27 have complementary or distinct functions in the melanosome and the obvious possibility that they bind distinct motor proteins has not escaped us. Other proteins identified by our analysis that are interesting to discuss include VDAC, and several subunits of vacuolar proton ATPases. Regulation of pH within melanosomes is an important regulatory factor for melanin production.52-55 The observation of melanosomes with aberrant pH in pinkeyeddilution melanocytes led Puri et al. to suggest that the p protein, a protein with 12 predicted membrane-spanning domains, is an ionic transporter responsible for maintaining low pH within melanocyte, which is critical for melanogenesis. In our early 78
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melanosome preparations, we were unable to detect the p protein by mass spectrometry, although obviously it might be delivered there at a later stage in melanosome maturation. To our knowledge, our study provides the first direct evidence of the presence of vATPases and VDAC, two pH regulators, which might modulate melanosomal pH and, thus, control rates of melanin biosynthesis. Finally, all six known melanosomal proteins serve as melanoma-specific immune targets56-58 and an interesting challenge is to determine if some of these novel melanosomal proteins can also serve that function. However, all studies using the known melanosomal proteins to elicit immune responses to interfere with the growth of tumors in melanoma patients have been encouraging but have not yet been significantly successful. The recent demonstration that flotillin,59 one of the novel melanosomal proteins identified in our study, is up-regulated in melanoma cells raises the possibility that these proteins might prove useful in eliciting immune responses in melanoma patients. Continuation of this work will be important to further resolve the patterns of expression of these novel melanosomal proteins in melanocytes and to elucidate their roles in melanosome structure and function. Complete characterization of the full repertoire of melanosomal proteins should resolve the unique structural and functional features of the melanosome, which is the ultimate goal of proteomics.
Acknowledgment. This work was supported in part by funding from USPHS GM 37537 (to D.F.H.).
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