bhf-ucl fitzgerald-2002-1 Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. 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 J. Biol. Chem. (0021-9258) 2002 imex curation Only protein-protein interactions 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 apoa1_human Apolipoprotein A-I APOA1 Apolipoprotein A1 protein protein human Homo sapiens Human MKAAVLTLAVLFLTGSQARHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ abca1-apoa1-6 1458063 n/a 1458064 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 false O95477:p.Cys1477Arg mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O95477:p.Gln597Arg mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O95477:p.Ser1506Leu mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O95477:p.Arg587Trp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false n/a 1458065 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 false physical association physical association false false false 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 1458063 n/a 1458064 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 false The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. n/a 1458065 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 false physical association physical association false false false 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 1458063 n/a 1458064 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 false The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. n/a 1458065 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 false physical association physical association false false false 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 1458063 n/a 1458064 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 false The sequence of the 8 amino acid flag tag is Asp-tyr-lys-asp-asp-asp-asp-lys. n/a 1458065 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 false physical association physical association false false false Figure 6 A