IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ lee-2012-1 Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). ecoli Escherichia coli (strain K12) pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Lee H.H., Nemecek D., Schindler C., Smith W.J., Ghirlando R., Steven A.C., Bonifacino J.S., Hurley J.H. hurley@helix.nih.gov J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation lee-2012-4 Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Lee H.H., Nemecek D., Schindler C., Smith W.J., Ghirlando R., Steven A.C., Bonifacino J.S., Hurley J.H. hurley@helix.nih.gov J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation lee-2012-2 Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). ecoli Escherichia coli (strain K12) electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Lee H.H., Nemecek D., Schindler C., Smith W.J., Ghirlando R., Steven A.C., Bonifacino J.S., Hurley J.H. hurley@helix.nih.gov J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation lee-2012-3 Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). ecoli Escherichia coli (strain K12) pull down pull down edman degradation edman degradation Lee H.H., Nemecek D., Schindler C., Smith W.J., Ghirlando R., Steven A.C., Bonifacino J.S., Hurley J.H. hurley@helix.nih.gov J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation lee-2012-5 Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). ecoli Escherichia coli (strain K12) enzymatic footprint enzymatic footprinting enzymatic footprint protein sequence protein sequence identification protein sequence Lee H.H., Nemecek D., Schindler C., Smith W.J., Ghirlando R., Steven A.C., Bonifacino J.S., Hurley J.H. Gel bands were excised and subjected to N-terminal amino acid sequencing using a 492 cLC protein sequencer (Applied Biosystems).The C-terminal boundaries of the fragments were inferred on the basis of MALDI-TOF mass spectra and the locations of potential Glu-C sites. hurley@helix.nih.gov J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation dtbp1_human Dysbindin My031 DTNBP1 Dystrobrevin-binding protein 1 Hermansky-Pudlak syndrome 7 protein Dysbindin-1 BLOC1S8 Biogenesis of lysosome-related organelles complex 1 subunit 8 protein protein human Homo sapiens Human MLETLRERLLSVQQDFTSGLKTLSDKSREAKVKSKPRTVPFLPKYSAGLELLSRYEDTWAALHRRAKDCASAGELVDSEVVMLSAHWEKKKTSLVELQEQLQQLPALIADLESMTANLTHLEASFEEVENNLLHLEDLCGQCELERCKHMQSQQLENYKKNKRKELETFKAELDAEHAQKVLEMEHTQQMKLKERQKFFEEAFQQDMEQYLSTGYLQIAERREPIGSMSSMEVNVDMLEQMDLMDISDQEALDVFLNSGGEENTVLSPALGPESSTCQNEITLQVPNPSELRAKPPSSSSTCTDSATRDISEGGESPVVQSDEEEVQVDTALATSHTDREATPDGGEDSDS bl1s4_human Biogenesis of lysosome-related organelles complex 1 subunit 4 BLOC1S4 CNO Protein cappuccino homolog protein protein human Homo sapiens Human MEGSFSDGGALPEGLAEEAEPQGAAWSGDSGTVSQSHSSASGPWEDEGAEDGAPGRDLPLLRRAAAGYAACLLPGAGARPEVEALDASLEDLLTRVDEFVGMLDMLRGDSSHVVSEGVPRIHAKAAEMRRIYSRIDRLEAFVRMVGGRVARMEEQVTKAEAELGTFPRAFKKLLHTMNVPSLFSKSAPSRPQQAGYEAPVLFRTEDYFPCCSERPQL bl1s5_human Biogenesis of lysosome-related organelles complex 1 subunit 5 BLOC1S5 MUTED Protein Muted homolog protein protein human Homo sapiens Human MSGGGTETPVGCEAAPGGGSKKRDSLGTAGSAHLIIKDLGEIHSRLLDHRPVIQGETRYFVKEFEEKRGLREMRVLENLKNMIHETNEHTLPKCRDTMRDSLSQVLQRLQAANDSVCRLQQREQERKKIHSDHLVASEKQHMLQWDNFMKEQPNKRAEVDEEHRKAMERLKEQYAEMEKDLAKFSTF snapn_human SNARE-associated protein Snapin SNAPIN SNAP25BP SNAPAP Synaptosomal-associated protein 25-binding protein BLOC1S7 Biogenesis of lysosome-related organelles complex 1 subunit 7 protein protein human Homo sapiens Human MAGAGSAAVSGAGTPVAGPTGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGIYPPGSPGK bl1s3_human Biogenesis of lysosome-related organelles complex 1 subunit 3 BLOC1S3 BLOS3 protein protein human Homo sapiens Human MASQGRRRRPLRRPETVVPGEATETDSERSASSSEEEELYLGPSGPTRGRPTGLRVAGEAAETDSEPEPEPEPTAAPRDLPPLVVQRESAEEAWGTEEAPAPAPARSLLQLRLAESQARLDHDVAAAVSGVYRRAGRDVAALASRLAAAQAAGLAAAHSVRLARGDLCALAERLDIVAGCRLLPDIRGVPGTEPEKDPGPRA bl1s2_human Biogenesis of lysosome-related organelles complex 1 subunit 2 BLOC1S2 BLOS2 CEAP Centrosome-associated protein protein protein human Homo sapiens Human MAAAAEGVLATRSDEPARDDAAVETAEEAKEPAEADITELCRDMFSKMATYLTGELTATSEDYKLLENMNKLTSLKYLEMKDIAINISRNLKDLNQKYAGLQPYLDQINVIEEQVAALEQAAYKLDAYSKKLEAKYKKLEKR bl1s1_human Biogenesis of lysosome-related organelles complex 1 subunit 1 BLOC1S1 BLOS1 RT14 GCN5L1 GCN5-like protein 1 Protein RT14 protein protein human Homo sapiens Human MAPGSRGERSSFRSRRGPGVPSPQPDVTMLSRLLKEHQAKQNERKELQEKRRREAITAATCLTEALVDHLNVGVAQAYMNQRKLDHEVKTLQVQAAQFAKQTGQWIGMVENFNQALKEIGDVENWARSIELDMRTIATALEYVYKGQLQSAPS bl1s6_human Biogenesis of lysosome-related organelles complex 1 subunit 6 BLOC1S6 PA PLDN Pallid protein homolog Pallidin Syntaxin 13-interacting protein protein protein human Homo sapiens Human MSVPGPSSPDGALTRPPYCLEAGEPTPGLSDTSPDEGLIEDLTIEDKAVEQLAEGLLSHYLPDLQRSKQALQELTQNQVVLLDTLEQEISKFKECHSMLDINALFAEAKHYHAKLVNIRKEMLMLHEKTSKLKKRALKLQQKRQKEELEREQQREKEFEREKQLTARPAKRM bloc1s1-bloc1s2-2 1298985 n/a 1298990 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1298991 unspecified role unspecified role prey prey n/a 1298992 unspecified role unspecified role prey prey n/a 1298993 unspecified role unspecified role prey prey n/a 1298994 unspecified role unspecified role prey prey n/a 1298995 unspecified role unspecified role prey prey n/a 1298996 unspecified role unspecified role prey prey n/a 1298997 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false association association false false false All eight subunits of BLOC-1 were coexpressed from a single polycistronic plasmid in E. coli. Coomassie staining showed no Ecoli proteins to co-purify with the 8 proteins. Fig. 1B dtnbp1-bloc1s1-3 1298985 n/a 1298997 unspecified role unspecified role bait bait n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n/a 1298991 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1298992 unspecified role unspecified role prey prey n/a 1298990 unspecified role unspecified role prey prey c-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1298994 unspecified role unspecified role prey prey n/a 1298993 unspecified role unspecified role prey prey n/a 1298995 unspecified role unspecified role prey prey n/a 1298996 unspecified role unspecified role prey prey association association false false false All eight subunits of BLOC-1 were coexpressed from a single polycistronic plasmid in E. coli. Coomassie staining showed no Ecoli proteins to co-purify with the 8 proteins. Fig. 3 pldn-bloc1s1-4 1298985 n/a 1298997 unspecified role unspecified role bait bait n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n/a 1298996 unspecified role unspecified role prey prey n/a 1298991 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false association association false false false Fig. 3 pldn-bloc1s1-6 1298985 n/a 1298990 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n/a 1298993 unspecified role unspecified role prey prey n/a 1298995 unspecified role unspecified role prey prey association association false false false Fig. 4A bloc1s2-dtnbp1-2 1298986 n/a 1298995 unspecified role unspecified role neutral component neutral component n/a 1298993 unspecified role unspecified role neutral component neutral component n/a 1298990 unspecified role unspecified role neutral component neutral component c-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 4B bloc1s2-dtnbp1-3 1298986 n/a 1298990 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1298995 unspecified role unspecified role neutral component neutral component n/a 1298993 unspecified role unspecified role neutral component neutral component physical association physical association false false false The dysbindin-Snapin-BLOS2 subcomplex eluted at a volume corresponding to a Stokes radius of 5.3 nm. Given the calculated molecular mass of 41.8 kDa for this construct, a Stokes radius of approx. 2.6 nm would have been expected for a hydrated globular protein of the same molecular mass. The actual value of 5.3 nm indicates that the complex is highly elongated. Fig. 4C bloc1s1-cno-1 1298986 n/a 1298996 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1298991 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1298997 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Fig. 6A, 6B The pallidin(33–160)-Cappuccino(1–177)-BLOS1(51–125) subcomplex was found to have a mass of 43.2 kDa and a Stokes radius of 4.6 nm dtnbp1-bloc1s1-2 1298987 n/a 1298992 unspecified role unspecified role neutral component neutral component n/a 1298996 unspecified role unspecified role neutral component neutral component n/a 1298995 unspecified role unspecified role neutral component neutral component n/a 1298994 unspecified role unspecified role neutral component neutral component n/a 1298993 unspecified role unspecified role neutral component neutral component n/a 1298990 unspecified role unspecified role neutral component neutral component n/a 1298991 unspecified role unspecified role neutral component neutral component n/a 1298997 unspecified role unspecified role neutral component neutral component physical association physical association false false false Subsequent analysis suggests BLOC-1 consists of eight globular domains of approximately equal diameter (30 A) connected in a linear chain. Fig. 2A pldn-bloc1s1-5 1298988 n/a 1298997 unspecified role unspecified role bait bait c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1298991 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1298996 unspecified role unspecified role prey prey association association false false false All eight subunits of BLOC-1 were coexpressed from a single polycistronic plasmid in E. coli. Coomassie staining showed no Ecoli proteins to co-purify with the 8 proteins. Fig. 3 bloc1s1-cno 1298989 n/a 1298996 unspecified role unspecified role neutral component neutral component n/a 1298991 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1298997 unspecified role unspecified role neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false association association false false false Fig. 5B