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/ Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. (0021-9258) 2012 imex curation 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 Only protein-protein interactions 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 Accepted 2012-JUN-27 by MGT J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. (0021-9258) 2012 imex curation 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 Only protein-protein interactions 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 Accepted 2012-JUN-27 by MGT J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. (0021-9258) 2012 imex curation 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 Only protein-protein interactions 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 Accepted 2012-JUN-27 by MGT J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. (0021-9258) 2012 imex curation 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 Only protein-protein interactions 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 Accepted 2012-JUN-27 by MGT J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. (0021-9258) 2012 imex curation 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 Only protein-protein interactions 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 Accepted 2012-JUN-27 by MGT 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 0504C86E12B66C08 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 3EA402AC53C81FFF bl1s5_human Biogenesis of lysosome-related organelles complex 1 subunit 5 BLOC1S5 MUTED Protein Muted homolog protein protein human Homo sapiens Human MSGGGTETPVGCEAAPGGGSKKRDSLGTAGSAHLIIKDLGEIHSRLLDHRPVIQGETRYFVKEFEEKRGLREMRVLENLKNMIHETNEHTLPKCRDTMRDSLSQVLQRLQAANDSVCRLQQREQERKKIHSDHLVASEKQHMLQWDNFMKEQPNKRAEVDEEHRKAMERLKEQYAEMEKDLAKFSTF 790D4DE8E97D83D1 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 B7D1FE64F242B25A bl1s2_human Biogenesis of lysosome-related organelles complex 1 subunit 2 BLOC1S2 BLOS2 CEAP Centrosome-associated protein protein protein human Homo sapiens Human MAAAAEGVLATRSDEPARDDAAVETAEEAKEPAEADITELCRDMFSKMATYLTGELTATSEDYKLLENMNKLTSLKYLEMKDIAINISRNLKDLNQKYAGLQPYLDQINVIEEQVAALEQAAYKLDAYSKKLEAKYKKLEKR 023DDBAC66E1912B bl1s3_human Biogenesis of lysosome-related organelles complex 1 subunit 3 BLOC1S3 BLOS3 protein protein human Homo sapiens Human MASQGRRRRPLRRPETVVPGEATETDSERSASSSEEEELYLGPSGPTRGRPTGLRVAGEAAETDSEPEPEPEPTAAPRDLPPLVVQRESAEEAWGTEEAPAPAPARSLLQLRLAESQARLDHDVAAAVSGVYRRAGRDVAALASRLAAAQAAGLAAAHSVRLARGDLCALAERLDIVAGCRLLPDIRGVPGTEPEKDPGPRA EB072B856AB6B4CF bl1s4_human Biogenesis of lysosome-related organelles complex 1 subunit 4 BLOC1S4 CNO Protein cappuccino homolog protein protein human Homo sapiens Human MEGSFSDGGALPEGLAEEAEPQGAAWSGDSGTVSQSHSSASGPWEDEGAEDGAPGRDLPLLRRAAAGYAACLLPGAGARPEVEALDASLEDLLTRVDEFVGMLDMLRGDSSHVVSEGVPRIHAKAAEMRRIYSRIDRLEAFVRMVGGRVARMEEQVTKAEAELGTFPRAFKKLLHTMNVPSLFSKSAPSRPQQAGYEAPVLFRTEDYFPCCSERPQL 46CFA7105DAF1071 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 37902FCFD4802294 bloc1s1-bloc1s2-2 1 6 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 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 12 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 11 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role prey prey 13 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 association association 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-bloc1s1-2 2 8 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component physical association physical association Subsequent analysis suggests BLOC-1 consists of eight globular domains of approximately equal diameter (30 A) connected in a linear chain. Fig. 2A dtnbp1-bloc1s1-3 1 13 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 12 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 8 unspecified role unspecified role prey prey 6 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 11 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role prey prey association association 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 1 13 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 9 unspecified role unspecified role prey prey 12 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 association association Fig. 3 pldn-bloc1s1-5 5 13 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 12 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 9 unspecified role unspecified role prey prey association association 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-bloc1s1-6 1 6 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 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 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey association association Fig. 4A bloc1s2-dtnbp1-2 4 10 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component 6 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig. 4B bloc1s2-dtnbp1-3 4 6 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 10 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association 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 3 9 unspecified role unspecified role neutral component neutral component 12 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 13 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 association association Fig. 5B bloc1s1-cno-1 4 9 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 ecoli Escherichia coli (strain K12) 12 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 ecoli Escherichia coli (strain K12) 13 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 ecoli Escherichia coli (strain K12) physical association physical association 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