MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. FEBS J. (1742-464X) 2014 imex curation John B.A., Meister M., Banning A., Tikkanen R. Ritva.Tikkanen@biochemie.med.uni-giessen.de 02-Apr-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Ritva.Tikkanen@biochemie.med.uni-giessen.de Accepted 2014-FEB-24 by LUANA 2014 imex curation Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. FEBS J. (1742-464X) 2014 imex curation John B.A., Meister M., Banning A., Tikkanen R. Ritva.Tikkanen@biochemie.med.uni-giessen.de 02-Apr-2014: Contacted by IntAct-Help. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Ritva.Tikkanen@biochemie.med.uni-giessen.de Accepted 2014-FEB-24 by LUANA 2014 imex curation Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. Flotillins bind to the Dileucine Sorting Motif of BACE1 and influence its endosomal Sorting. FEBS J. (1742-464X) 2014 imex curation John B.A., Meister M., Banning A., Tikkanen R. Ritva.Tikkanen@biochemie.med.uni-giessen.de 02-Apr-2014: Contacted by IntAct-Help. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 pla proximity ligation assay pELISA pELISA PLA immunostaining immunostaining Ritva.Tikkanen@biochemie.med.uni-giessen.de Accepted 2014-FEB-24 by LUANA 2014 imex curation flot2_human Flotillin-2 FLOT2 ESA1 M17S1 Epidermal surface antigen Membrane component chromosome 17 surface marker 1 protein protein human Homo sapiens Human MGNCHTVGPNEALVVSGGCCGSDYKQYVFGGWAWAWWCISDTQRISLEIMTLQPRCEDVETAEGVALTVTGVAQVKIMTEKELLAVACEQFLGKNVQDIKNVVLQTLEGHLRSILGTLTVEQIYQDRDQFAKLVREVAAPDVGRMGIEILSFTIKDVYDKVDYLSSLGKTQTAVVQRDADIGVAEAERDAGIREAECKKEMLDVKFMADTKIADSKRAFELQKSAFSEEVNIKTAEAQLAYELQGAREQQKIRQEEIEIEVVQRKKQIAVEAQEILRTDKELIATVRRPAEAEAHRIQQIAEGEKVKQVLLAQAEAEKIRKIGEAEAAVIEAMGKAEAERMKLKAEAYQKYGDAAKMALVLEALPQIAAKIAAPLTKVDEIVVLSGDNSKVTSEVNRLLAELPASVHALTGVDLSKIPLIKKATGVQV F690BFE0151D3BD7 flot1_rat Flotillin-1 Flot1 Reg2 Reggie-2 protein protein rat Rattus norvegicus (Rat) Rat MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEKVYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQRAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHSLGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQCLSEIEMAKAQRDYELKKATYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQEQEIARREKELEARVRKPAEAERYRLERLAEAEKAQLIMQAEAEAESVRMRGEAEAFAVGARARAEAEQMAKKAEAFQMYQEAAQLDMLLEKLPQVAEEISGPLTSANKITLVSSGSGTMGAAKVTGEVLDILSRLPESVERLTGVSISQVNHNKPLRTA B3B54DDB81705EAF bace1_human Beta-secretase 1 BACE KIAA1149 Beta-site amyloid precursor protein cleaving enzyme 1 Membrane-associated aspartic protease 2 Memapsin-2 Aspartyl protease 2 BACE1 protein protein human Homo sapiens Human MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLRLPRETDEEPEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWRCLRCLRQQHDDFADDISLLK DIP protein P56817 original sequence version: 161 377CE4C824ACEF05 377CE4C824ACEF05 flot1_human Flotillin-1 FLOT1 protein protein human Homo sapiens Human MFFTCGPNEAMVVSGFCRSPPVMVAGGRVFVLPCIQQIQRISLNTLTLNVKSEKVYTRHGVPISVTGIAQVKIQGQNKEMLAAACQMFLGKTEAEIAHIALETLEGHQRAIMAHMTVEEIYKDRQKFSEQVFKVASSDLVNMGISVVSYTLKDIHDDQDYLHSLGKARTAQVQKDARIGEAEAKRDAGIREAKAKQEKVSAQYLSEIEMAKAQRDYELKKAAYDIEVNTRRAQADLAYQLQVAKTKQQIEEQRVQVQVVERAQQVAVQEQEIARREKELEARVRKPAEAERYKLERLAEAEKSQLIMQAEAEAASVRMRGEAEAFAIGARARAEAEQMAKKAEAFQLYQEAAQLDMLLEKLPQVAEEISGPLTSANKITLVSSGSGTMGAAKVTGEVLDILTRLPESVERLTGVSISQVNHKPLRTA C69E5421E565F53A bace1-flot1 1 flotillin-1 5 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey purified purified BACE1 6 predetermined predetermined participant predetermined western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain P56817:p.Leu499_Leu500delinsAlaAla mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain LL AA Thu Jul 07 12:15:40 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was leu499ala_leu500ala Fri Jul 22 10:52:37 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'leu499ala,leu500ala' Wed May 02 15:55:17 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu_leu499-500ala_ala' Tue Jun 30 19:50:14 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu499_Leu500delinsAlaAla' direct interaction direct interaction f1d To dissect the role of flotillins in BACE1 binding in more detail and to test if the interaction is a direct one, we performed direct pulldown assays using bacterially expressed, purified proteins (Figure 1D). Purified flotillin-1 again strongly bound to the WT BACE1-GST tail, whereas no binding was observed with the LLAA tail. Only a very weak binding that was close to the GST background level was observed with purified flotillin-2. Taken together, these data show that flotillin-1 directly interacts with the cytoplasmic tail of BACE1 and that this interaction is mediated by the well known dileucine sorting motif in BACE1 flot2-bace1 2 flotillin-2 4 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level BACE1 6 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2a To corroborate these findings in an in vivo context, we performed coimmunoprecipitation experiments with endogenous flotillins and BACE1 (Figure 2A). For this, we used polyclonal flotillin-2 antibodies since they result in a quantitative precipitation of the pool of flotillin-1/flotillin-2 complexes in the cells. BACE1 was found to be immunoprecipitated with endogenous flotillin-2, verifying the relevance of the pulldown data. bace1-flot2 3 flotillin-2 4 immunostaining immunostaining immunostaining immunostaining unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell BACE1 6 immunostaining immunostaining immunostaining immunostaining unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell proximity proximity close-contact colocalization neighbourhood interaction f2b To dissect the role of flotillins in BACE1 binding in more detail and to test if the interaction is a direct one, we performed direct pulldown assays using bacterially expressed, purified proteins (Figure 1D). Purified flotillin-1 again strongly bound to the WT BACE1-GST tail, whereas no binding was observed with the LLAA tail. Only a very weak binding that was close to the GST background level was observed with purified flotillin-2. Taken together, these data show that flotillin-1 directly interacts with the cytoplasmic tail of BACE1 and that this interaction is mediated by the well known dileucine sorting motif in BACE1. To corroborate these findings in an in vivo context, we performed coimmunoprecipitation experiments with endogenous flotillins and BACE1 (Figure 2A). For this, we used polyclonal flotillin-2 antibodies since they result in a quantitative precipitation of the pool of flotillin-1/flotillin-2 complexes in the cells. BACE1 was found to be immunoprecipitated with endogenous flotillin-2, verifying the relevance of the pulldown data. Since the signals observed in the coprecipitation experiment were very weak, we used the Proximity Ligation Assay (PLA) to verify the interaction of endogenous BACE1 and flotillins. PLA is based on the use of specific antibodies and can detect two proteins that are at very close vicinity (less than 40 nm) in cells and is generally used to study interaction of proteins [38-40]. Interacting proteins are visualized as fluorescent signals whose localization in the cells can also be assessed, giving information on the compartment in which the interaction takes place. However, the signal intensities as such do not correlate with the strength or quantity of the interaction. Quantification of the signals is done by counting the amount of fluorescent signals per cells and comparing these to negative controls (performed without the first antibody). PLA analysis of endogenous flotillin-1 or flotillin-2 and BACE1 showed that the fluorescent dots that arise upon interaction of these proteins were mainly detected in the perinuclear region of the cells, with few dots residing in the cell periphery (Fig. 2B). bace1-flot2-1 3 flotillin-1 7 immunostaining immunostaining immunostaining immunostaining unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell BACE1 6 immunostaining immunostaining immunostaining immunostaining unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell proximity proximity close-contact colocalization neighbourhood interaction f2b To dissect the role of flotillins in BACE1 binding in more detail and to test if the interaction is a direct one, we performed direct pulldown assays using bacterially expressed, purified proteins (Figure 1D). Purified flotillin-1 again strongly bound to the WT BACE1-GST tail, whereas no binding was observed with the LLAA tail. Only a very weak binding that was close to the GST background level was observed with purified flotillin-2. Taken together, these data show that flotillin-1 directly interacts with the cytoplasmic tail of BACE1 and that this interaction is mediated by the well known dileucine sorting motif in BACE1. To corroborate these findings in an in vivo context, we performed coimmunoprecipitation experiments with endogenous flotillins and BACE1 (Figure 2A). For this, we used polyclonal flotillin-2 antibodies since they result in a quantitative precipitation of the pool of flotillin-1/flotillin-2 complexes in the cells. BACE1 was found to be immunoprecipitated with endogenous flotillin-2, verifying the relevance of the pulldown data. Since the signals observed in the coprecipitation experiment were very weak, we used the Proximity Ligation Assay (PLA) to verify the interaction of endogenous BACE1 and flotillins. PLA is based on the use of specific antibodies and can detect two proteins that are at very close vicinity (less than 40 nm) in cells and is generally used to study interaction of proteins [38-40]. Interacting proteins are visualized as fluorescent signals whose localization in the cells can also be assessed, giving information on the compartment in which the interaction takes place. However, the signal intensities as such do not correlate with the strength or quantity of the interaction. Quantification of the signals is done by counting the amount of fluorescent signals per cells and comparing these to negative controls (performed without the first antibody). PLA analysis of endogenous flotillin-1 or flotillin-2 and BACE1 showed that the fluorescent dots that arise upon interaction of these proteins were mainly detected in the perinuclear region of the cells, with few dots residing in the cell periphery (Fig. 2B).