Table I

Proteins identified in NM granules by LC-MS/MS analysis

E, endosome; ECM, extra cellular matrix; Ly, lysosome; Mt, mitochondrion; PM, plasma membrane; V-ATPase, vacuolar ATPase; UCC1, up-regulated in colorectal cancer gene-1; CALM, clathrin assembly lymphoid myeloid leukemia protein; HSP, heat shock protein; MAP, mitogen-activated protein; GM2, GalNAcβ 1→ 4(Neu5Acα 2→ 3)Galβ 1→ 4Glcβ 1→ 1′-ceramide; LPIC, lysosomal pepstatin-insensitive carboxypeptidase.

Protein no. and nameSwiss-Prot accession numberMolecular mass[Peptides]2+ sequenced; sequence coverageLocationComments
kDa
Lysosomal membrane
    1 CD63 antigen (LAMP-3) P08962 25.61; 4.2%LyIntegral membrane protein; ocular melanoma-associated antigen
    2 LIMP II Q14108 54.33; 20.9%LyType II membrane protein; may act as a lysosomal receptor
    3 LAMP-1 (CD107a) P11279 41.62; 7.1%Ly, ELysosomal and endosomal membrane
    4 V-ATPase, subunit A1 P38606 68.22; 5.2%LyAcidification of intracellular compartments
    5 V-ATPase, subunit B2 P21281 58.43; 14.8%LyAcidification of intracellular compartments
    6 V-ATPase, subunit E P36543 26.22; 12.0%LyAcidification of intracellular compartments
Proteases
    7 Cathepsin B P07858 28.810; 43.3%LyThiol protease; peptidase family C1
    8 Cathepsin D P07339 44.610; 34.2%LyAspartyl protease; peptidase A1
    9a Cathepsin Z (contains cathepsins X and Z) Q9UBR 33.82; 7.9%LyPeptidase family C1
    10 Tripeptidyl-peptidase I (TPP-I; LPIC) O14773 61.25; 16.3%LyLysosomal serine protease; peptidase family S53; ceroid lipofuscinosis, neuronal 2, late infantile (CLN2)
    11 γ-Glutamyl hydrolase Q92820 35.911; 52.1%LyLysosomal or secreted; peptidase family C26
    12 Lysosomal Pro-X carboxypeptidase (angiotensinase C) P42785 55.83; 9.0%Serine carboxypeptidase; peptidase family S28
    13 Ubiquitin carboxyl-terminal hydrolase 5 (isopeptidase T) Q96J22 93.22; 5.3%Ly?Zinc-containing ubiquitin specific protease; peptidase family C19
    14 Dipeptidyl-peptidase II (DPP-II) Q9UHL4 54.34; 17.8%LyLysosomal and intracellular vesicles; peptidase family S28
(Glyco)lipid metabolism
    15 Acid ceramidase (acylsphingosine deacylase) Q13510 44.614; 46.2%LyHydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid; Farber lipogranulomatosis
    16 Phospholipase D3 Q8IV08 54.77; 19.7%?
    17 Palmitoyl protein thioesterase 1 P50897 34.25; 34.2%LyRemoval of thioester-linked fatty acyl groups from modified cysteine residues in proteins; ceroid lipofuscinosis, neuronal 1, infantile (CLN1) and neuronal 4, adult type (CLN4)
    18 β-Hexosaminidase (N-acetyl-β-glucosaminidase) P07686 63.14; 17.7%LyDegradation of GM2 gangliosides; Sandhoff disease
    19 Galactosylceramidase P54803 75.12; 4.9%LyLysosomal catabolism of galactosylceramide, a major lipid in myelin; Krabbe disease
    20 Proactivator polypeptide (cerebroside sulfate activator protein) P07602 58.53; 6.1%LyContains saposins A, B, C, D; lipid binding and transport; glycosphingolipid metabolism; lysosome organization and biogenesis
Glycoprotein metabolism
    21 Arylsulfatase B (ARSB) P15848 59.71; 3.8%LyDegradation of chondroitin sulfate and dermatan sulfate; mucopolysaccharidosis type VI (MPS-VI, Maroteaux-Lamy syndrome)
    22 N-Acetylgalactosamine-6-sulfatase (chondroitinase) P34059 58.04; 16.8%LyDegradation of chondroitin sulfate and dermatan sulfate; mucopolysaccharidosis type IVA (MPS-IVA, Morquio A syndrome)
    23a N-Sulfoglucosamine sulfohydrolase (sulfaminase) P51688 56.73; 13.9%LyDegradation of heparan sulfate; mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome A)
    24 N4-(β-N-Acetylglucosaminyl)-l-asparaginase (AGA) P20933 37.31; 9.0%LyGlycoprotein catabolism; cleaves the GlcNAc-Asn bond
    25 Lysosomal α-glucosidase P10253 105.31; 1.9%LyEssential for degradation of glycogen in lysosomes; Pompe disease
Unknown
    26 High mobility group protein 1 P09429 24.79; 49.8%?Secreted or nuclear (73); gastrointestinal stromal tumor marker (74)
    27 R31109_1 (25-kDa protein; similar to high mobility group 20B) Q9Y4A2 24.63; 22.5%?Similar to high mobility group protein 1
    28a Hypothetical protein LOC196463 Q8NHP8 65.53;?
    29 25-kDa brain-specific protein O94811 23.713; 57.7%?Brain-specific expression; glycogen synthase kinase 3 (GSK3) inhibitor p24; p25 could act as a unique MAP in vivo (75)
Probable cytosolic proteins
    30 Mammalian ependymin-related protein-1 (MERP-1; UCC1 protein) Q9UM22 19.95; 55.0%?Secreted
    31 Tyrosine 3-monooxygenase (tyrosine 3-hydroxylase) P07101 58.53; 12.3%CytosolCatalyzes the first step in catecholamine biosynthesis; putatively involved in the synthesis of neuromelanin (76)
    32 14-3-3 protein ζ/δ P29312 27.72; 12.7%CytosolActivates tyrosine 3-hydroxylase
    33 14-3-3 Protein γ P35214 28.33; 13.4%CytosolActivates tyrosine 3-hydroxylase
    34a Peroxiredoxin 1 Q06830 22.13; 15.5%Ly, cytosolAntioxidant protection
    35 Heat shock cognate 71-kDa protein (heat shock 70-kDa protein 8) P11142 53.54; 10.4%CytosolChaperone, protein folding; ATPase activity; maybe functions as an endogenous inhibitory regulator of hsc70
    36 α-Crystallin B chain P02511 22.411; 76.8%Accumulates in the brains of patients with Alexander disease; HSP20 family
    37 Heat shock 70-kDa protein 9B (mortalin-2) Q8N1C8 73.73; 7.4%?Calcium and ATP binding; modulates toxicity of α-synuclein (77)
    38 Heat shock protein 90-α P07900 84.65; 7.5%CytosolATPase activity (by similarity)
    39 Glycogen phosphorylase, brain form P11216 96.75; 9.2%CytosolCarbohydrate metabolism
    40 6-Phosphofructokinase P78457 85.05; 9.9%CytosolGlycolysis
    41 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) P04406 35.92; 9.6%CytosolGlycolysis
Others
    42 Ubiquitin P02248 8.6Proteasome- or lysosome-based protein degradation; protein transport to lysosomes
    43 Similar to cytosolic sialic acid 9-O-acetylesterase homolog Q8IUT9 58.111; 38.8%Ly, E9-O-Acetylation of sialic acids, localized in lysosomes and endosomes
    44 Myelin basic protein P02686 21.517; 61.9%
    45a Myelin proteolipid protein (PLP) P60201 30.12; 8.7%Integral membrane protein; Pelizaeus-Merzbacher disease
    46 2′,3′-Cyclic nucleotide 3′-phosphodiesterase P09543 47.68; 20.7%Bound to membrane structures of brain white matter, myelin-associated
    47a Sodium/potassium-transporting ATPase, α-3 chain P13637 111.73; 6.0%PM, ESodium/potassium exchange; probably regulated by endocytosis (57)
    48a Voltage-dependent anion-selective channel P (VDAC 1) P21796 30.63; 18.1%Mt, E, LyAllows diffusion of small hydrophilic molecules
    49a Apolipoprotein D (apoD) P05090 21.25; 21.2%Endocytosed; lipid metabolism
    50 Cystatin C P01034 15.84; 40.9%Cysteine protease inhibitor; amyloidogenic properties
Mitochondrial proteins
    51 Similar to ATP synthase, mitochondrial F1 complex, α subunit Q96FB4 59.84; 14.5%Mt, LyF1 ATPase subunit
    52 ATP synthase β chain P06576 56.512; 42.9%MtF1 ATPase subunit
    53a ATP synthase D chain O5947 18.41; 12.4%MtF1 ATPase subunit
    54a ATP synthase g chain O75964 11.41; 14.6%Mt, LyF1 ATPase subunit
    55 60-kDa heat shock protein (HSP60) P10809 61.12; 7.7%MtMitochondrial matrix; protein folding
    56 Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) P36969 22.12; 13.2%Mt, cytosolProtects against oxidative damage and the toxicity of ingested lipid hydroperoxides
Vesicular traffic
    57 Tubulin α 6 Q9BQE3 49.94; 20.8%Major constituent of microtubules
    58 Tubulin β 2 P05217 49.85;19.8%Major constituent of microtubules
    59 Microtubule-associated protein 2 P11137 202.811;10.0%May stabilize the microtubules against depolymerization
    60 Microtubule-associated protein tau P10636 78.72; 6.1%Microtubule assembly
    61 Clathrin heavy chain Q00610 191.61; 1.1%Cytoplasmic face of coated pits and vesicles
    62 Phosphatidylinositol-binding clathrin assembly protein (CALM, AP180-2) Q13492 70.71; 3.4%Assembly of clathrin, clathrin coated-vesicle formation; membrane recycling
    63a Phosphatidylinositol 4-phosphate 5-kinase type II α P48426 46.22; 8.1%E, LyAssociated with membranes of the late endocytic pathway
    64a α-SNAP P54920 3; 14.2%VesiclesParticipates in various endocytotic events and vesicular traffic
    65a Syntaxin-binding protein 1 (Hunc18) P61764 67.63; 7.9%VesiclesParticipates in various endocytotic events and vesicular traffic
    66 Dynamin-1, neural isoform Q05193 97.43; 3.5%E, vesiclesParticipates in various endocytotic events and vesicular traffic
    67 NipSnap1 protein Q9BPW8 33.33; 16.6%Vesicles?Putative involvement in vesicular traffic
    68 NipSnap2 protein O75323 33.84; 31.8%Vesicles?Transmembrane domain; putative involvement in vesicular traffic
Endoplasmic reticulum
    69 Calreticulin P27797 46.82; 7.2%ER, cytosol, ECMChaperone promoting folding, assembly, and quality control in the ER via the calreticulin/calnexin cycle; associated with the lytic granules
    70 Calnexin (p90) P27824 67.64; 9.8%ERType I membrane protein; melanogenetic chaperone; not associated to classical lysosomes but to some lysosome-related organelles
    71 Dolichyl-diphosphooligosac-charide-protein glyco-syltransferase (ribophorin I) P04843 68.63; 7.1%ERType I membrane protein; N-oligosaccharyltrans-ferase enzyme (transfer of a high mannose oligosaccharide from a lipid-linked donor to an asparagine residue)
    72 Protein-disulfide isomerase A3 (ERp57) P30101 54.34; 12.0%ERProtein-disulfide isomerase family
  • a These proteins were not identified in all five neuromelanin granule preparations.