bhf-ucl Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J. Biol. Chem. (0021-9258) 2002 imex curation Fitzgerald M.L., Morris A.L., Rhee J.S., Andersson L.P., Mendez A.J., Freeman M.W. Cardiac - Interactions involving cardiac related proteins The source of ABCA1 constructs is human as per author citation to own previous paper (reference 17) PMID 11328826. The source of ApoA-I is purified human as per author citation to paper PMID 10918065 (reference 13). freeman@molbio.mgh.harvard.edu 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions human-293 ebna Homo sapiens 293 ebna 293 ebna crosslink cross-linking study crosslink weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Fitzgerald M.L., Morris A.L., Rhee J.S., Andersson L.P., Mendez A.J., Freeman M.W. Cardiac - Interactions involving cardiac related proteins The author cites reference 13 (PMID 10918065) for cross-linking method. The reference states that the cells were first incubated with purififed I125-ApoA-1. The bifunctional cross-linker DSP was then added thus cross-linking Apoa-I and ABCA1. This was followed by cell lysis, anti-bait Co-IP of the complex with an anti-ABCA1 antibody, SDS-PAGE and radiography. The source of ABCA1 constructs is human as per author citation to own previous paper (reference 17) PMID 11328826. The source of ApoA-I is purified human as per author citation to paper PMID 10918065 (reference 13). freeman@molbio.mgh.harvard.edu Accepted 2013-DEC-09 by ORCHARD J. Biol. Chem. (0021-9258) 2002 imex curation abca1_human Phospholipid-transporting ATPase ABCA1 ATP-binding cassette transporter 1 Cholesterol efflux regulatory protein ABCA1 ABC1 CERP ATP-binding cassette sub-family A member 1 protein protein human Homo sapiens Human MACWPQLRLLLWKNLTFRRRQTCQLLLEVAWPLFIFLILISVRLSYPPYEQHECHFPNKAMPSAGTLPWVQGIICNANNPCFRYPTPGEAPGVVGNFNKSIVARLFSDARRLLLYSQKDTSMKDMRKVLRTLQQIKKSSSNLKLQDFLVDNETFSGFLYHNLSLPKSTVDKMLRADVILHKVFLQGYQLHLTSLCNGSKSEEMIQLGDQEVSELCGLPREKLAAAERVLRSNMDILKPILRTLNSTSPFPSKELAEATKTLLHSLGTLAQELFSMRSWSDMRQEVMFLTNVNSSSSSTQIYQAVSRIVCGHPEGGGLKIKSLNWYEDNNYKALFGGNGTEEDAETFYDNSTTPYCNDLMKNLESSPLSRIIWKALKPLLVGKILYTPDTPATRQVMAEVNKTFQELAVFHDLEGMWEELSPKIWTFMENSQEMDLVRMLLDSRDNDHFWEQQLDGLDWTAQDIVAFLAKHPEDVQSSNGSVYTWREAFNETNQAIRTISRFMECVNLNKLEPIATEVWLINKSMELLDERKFWAGIVFTGITPGSIELPHHVKYKIRMDIDNVERTNKIKDGYWDPGPRADPFEDMRYVWGGFAYLQDVVEQAIIRVLTGTEKKTGVYMQQMPYPCYVDDIFLRVMSRSMPLFMTLAWIYSVAVIIKGIVYEKEARLKETMRIMGLDNSILWFSWFISSLIPLLVSAGLLVVILKLGNLLPYSDPSVVFVFLSVFAVVTILQCFLISTLFSRANLAAACGGIIYFTLYLPYVLCVAWQDYVGFTLKIFASLLSPVAFGFGCEYFALFEEQGIGVQWDNLFESPVEEDGFNLTTSVSMMLFDTFLYGVMTWYIEAVFPGQYGIPRPWYFPCTKSYWFGEESDEKSHPGSNQKRISEICMEEEPTHLKLGVSIQNLVKVYRDGMKVAVDGLALNFYEGQITSFLGHNGAGKTTTMSILTGLFPPTSGTAYILGKDIRSEMSTIRQNLGVCPQHNVLFDMLTVEEHIWFYARLKGLSEKHVKAEMEQMALDVGLPSSKLKSKTSQLSGGMQRKLSVALAFVGGSKVVILDEPTAGVDPYSRRGIWELLLKYRQGRTIILSTHHMDEADVLGDRIAIISHGKLCCVGSSLFLKNQLGTGYYLTLVKKDVESSLSSCRNSSSTVSYLKKEDSVSQSSSDAGLGSDHESDTLTIDVSAISNLIRKHVSEARLVEDIGHELTYVLPYEAAKEGAFVELFHEIDDRLSDLGISSYGISETTLEEIFLKVAEESGVDAETSDGTLPARRNRRAFGDKQSCLRPFTEDDAADPNDSDIDPESRETDLLSGMDGKGSYQVKGWKLTQQQFVALLWKRLLIARRSRKGFFAQIVLPAVFVCIALVFSLIVPPFGKYPSLELQPWMYNEQYTFVSNDAPEDTGTLELLNALTKDPGFGTRCMEGNPIPDTPCQAGEEEWTTAPVPQTIMDLFQNGNWTMQNPSPACQCSSDKIKKMLPVCPPGAGGLPPPQRKQNTADILQDLTGRNISDYLVKTYVQIIAKSLKNKIWVNEFRYGGFSLGVSNTQALPPSQEVNDAIKQMKKHLKLAKDSSADRFLNSLGRFMTGLDTKNNVKVWFNNKGWHAISSFLNVINNAILRANLQKGENPSHYGITAFNHPLNLTKQQLSEVALMTTSVDVLVSICVIFAMSFVPASFVVFLIQERVSKAKHLQFISGVKPVIYWLSNFVWDMCNYVVPATLVIIIFICFQQKSYVSSTNLPVLALLLLLYGWSITPLMYPASFVFKIPSTAYVVLTSVNLFIGINGSVATFVLELFTDNKLNNINDILKSVFLIFPHFCLGRGLIDMVKNQAMADALERFGENRFVSPLSWDLVGRNLFAMAVEGVVFFLITVLIQYRFFIRPRPVNAKLSPLNDEDEDVRRERQRILDGGGQNDILEIKELTKIYRRKRKPAVDRICVGIPPGECFGLLGVNGAGKSSTFKMLTGDTTVTRGDAFLNKNSILSNIHEVHQNMGYCPQFDAITELLTGREHVEFFALLRGVPEKEVGKVGEWAIRKLGLVKYGEKYAGNYSGGNKRKLSTAMALIGGPPVVFLDEPTTGMDPKARRFLWNCALSVVKEGRSVVLTSHSMEECEALCTRMAIMVNGRFRCLGSVQHLKNRFGDGYTIVVRIAGSNPDLKPVQDFFGLAFPGSVLKEKHRNMLQYQLPSSLSSLARIFSILSQSKKRLHIEDYSVSQTTLDQVFVNFAKDQSDDDHLKDLSLHKNQTVVDVAVLTSFLQDEKVKESYV DIP protein O95477 original sequence version: 70 DIP protein O95477 original sequence version: 169 21A2CF8F3F518D6D apoa1_human Apolipoprotein A-I APOA1 Apolipoprotein A1 protein protein human Homo sapiens Human MKAAVLTLAVLFLTGSQARHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ DIP protein P02647 original sequence version: 116 DIP protein P02647 original sequence version: 125 DIP protein P02647 original sequence version: 147 DIP protein P02647 original sequence version: 162 1A28B8366E620310 abca1-apoa1-6 1 2 unspecified role unspecified role neutral component neutral component cell lysate cell lysate O95477:p.Trp590Ser mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain W S Wed May 02 15:08:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'trp590ser' Tue Jun 30 15:56:34 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Trp590Ser' O95477:p.Cys1477Arg mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain C R Wed May 02 15:31:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys1477arg' Tue Jun 30 16:40:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys1477Arg' O95477:p.Gln597Arg mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain Q R Wed May 02 15:38:21 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln597arg' Tue Jun 30 19:17:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln597Arg' O95477:p.Ser1506Leu mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain S L Wed May 02 16:01:41 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser1506leu' Tue Jun 30 16:49:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser1506Leu' O95477:p.Arg587Trp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain R W Wed May 02 16:01:33 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg587trp' Tue Jun 30 16:29:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg587Trp' 3 unspecified role unspecified role neutral component neutral component purified purified 125i radiolabel 125i radiolabel 125i radiolabel I125 125I 125I I125 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 4C, 6A, 7A, 8 Under reducing conditions, using the bifunctional cross-linking agent DSP (figure 4 C, 6 A and 7A) or DSTTP (figure 7 A), resulted in ApoA-I becoming detached from ABCA1.This is because DSP and DSTTP cross-linkers contain thiol-thiol sensitive bonds. However, using DSG cross-linker (figure 7 A), ApoA-I becomes detached as DSG does not contain thiol-sensitive bonds. Figure 7 B: Cross-linking efficiency using DSP cross-linker has a linear relationship with temperature. The efficiency is highest at 37 degrees centigrade, intermediate at 25 and drops down significantly at 4. abca1-apoa1-5 1 2 unspecified role unspecified role neutral component neutral component cell lysate cell lysate flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged certain certain sequence position certain certain certain sequence position certain The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. 3 unspecified role unspecified role neutral component neutral component purified purified 125i radiolabel 125i radiolabel 125i radiolabel I125 125I 125I I125 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 4 C Author states that the flag tag at amino acid 207 causes a modest increase the binding strength between ApoA-1 and ABCA1 as shown by the darker band on the gel. abca1-apoa1-7 1 2 unspecified role unspecified role neutral component neutral component cell lysate cell lysate flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged certain certain sequence position certain certain certain sequence position certain The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. 3 unspecified role unspecified role neutral component neutral component purified purified 125i radiolabel 125i radiolabel 125i radiolabel I125 125I 125I I125 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 4 C Author states that the flag tag at amino acid 1396 does not affect the binding strength between ApoA-1 and ABCA1. abca1-apoa1-8 1 2 unspecified role unspecified role neutral component neutral component cell lysate cell lysate rhodopsin epitope tag c-terminus tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. 3 unspecified role unspecified role neutral component neutral component purified purified 125i radiolabel 125i radiolabel 125i radiolabel I125 125I 125I I125 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 6 A