MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url&gt;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 Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. FEBS Lett. (0014-5793) 2012 Wiemuth D., van de Sandt L., Herr R., Gründer S. pig-cl4 Sus scrofa cl4 cl4 fluorescence imaging fluorescence microscopy fluorescence imaging tag fluorescence tag visualisation by fluorescence tag fluorescence Wiemuth D., van de Sandt L., Herr R., Gründer S. FEBS Lett. (0014-5793) 2012 Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. FEBS Lett. (0014-5793) 2012 Wiemuth D., van de Sandt L., Herr R., Gründer S. yeast Saccharomyces cerevisiae Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid experimental particp experimental participant identification experimental particp Wiemuth D., van de Sandt L., Herr R., Gründer S. FEBS Lett. (0014-5793) 2012 Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. Transient receptor potential N (TRPN1) from Xenopus interacts with the penta-EF-hand protein peflin. FEBS Lett. (0014-5793) 2012 Wiemuth D., van de Sandt L., Herr R., Gründer S. in vitro In vitro pull down pull down experimental particp experimental participant identification experimental particp Wiemuth D., van de Sandt L., Herr R., Gründer S. FEBS Lett. (0014-5793) 2012 pef1_xenla Peflin pef1 PEF protein with a long N-terminal hydrophobic domain Penta-EF hand domain-containing protein 1 protein protein xenla Xenopus laevis (African clawed frog) African clawed frog MASYPYGQGYHGAAGQPPGAPQTNYYGGQQYGGGVQPAASYGRPAPGAPYGSPPSGGVYGHPVPGSAAPGAPGGPYGGQAPGGPYSVPGSTPYGSQQHGSYGQGAPAGNIPPGVDPEAFSWFQTVDTDHSGYISLKELKQALVNTNWSSFNDETCTMMMNMFDKSNSGRIDMFGFSALWRFIQQWRNLFQQYDRDRSGSINQGELHQALCQMGYQLSPQFVQIVMSRYAQRSAQPGLQLDRFIQICTQLQSMTEAFREKDTGQIGTAKLSYEDFITMTTTRLL mint 129F451E202CBE1F q4a520_xenla XB5813854.L XB5813854 XB5813854.L [provisional:ankrd52] nompC protein protein xenla Xenopus laevis (African clawed frog) African clawed frog MSLQDCLTALHVAAAHSKDEIVRLLLKKAEPNIPGGPKDQLPLHYTALRPNGAINVTQTLLRASHKDARLTPDKDGCIPLFLAIEAGNLGICKELLLVNPESQLRAVSKKYGDTALHASCRKRDVDAAKLLVEYGANVDCQNDEGQTPLHIVAWAGDEMMLKFLHQCKTNANITDKMERTPLHVAAERGNTNVVEILTEKFRSNVLARTKDGNTLMHIASQCGHPETALAFLKRGVLLHMPNKSGAVCLHAAAKRGHTAVVKALLQKGAHVDARTKDNYTALHVAAENCKPQVVQTLLGFGAQVQLKGGKAQETPLHVAARIKDGEKVAEMLIKSGADINVEQENGETAMHISAQHGNLKMITALIEEGGEPTWQSKNQENPLHTAVRHCHLPIVEVLLQHLINERGRADAVGCVNQANKAGETSLHLAAAVKKEMIHTKEEDVKIIGILMEHDGDISRPTNTTLETPLHYCARVGNEDVLLEMIRHISSSRMQQTMNKQAKNGRSPLLVAAEQGHTGIVRILLQNQARVDVFDEHGKAALHLAAENGHDKIADILLKHKAFVNAKTKLGLTPLHLCAQNGFNHLVKLLVGTHSASIDAMALTKRTPLHMAALNGQLDVCNSLLNMKADVNATDIEGQTPLHLAAENDHSEVVKVFLKHKPELVTSANMEGSTCAHIAASKGSAAVIKELLKFNKTGVTTARNKTNDSTPLHLAAAGGHTDVVKVLLETGALASDENGEGMTAIHLAAKNGHINVLEALKGSVSFRITSTKTGFTALHVAAHFGQLDFVREILTKVPATMTSEPPKAVPDLLHMKEQRGESGYTPLHLASQSGHESLVRLLLNYPGVQADTATTRQGSTPIHLAAQNGHTAVVGLLLSKSTSQLHMKDKRGRTCLHLAAANGHIEMMRALIGQGAEINVTDKNGWCPLHFAARSGFLDTVRFLVECGANPTLECKDGKTAIQYAAAKNHQDVVSFLLKKNHNTLKLIEDRKFVFDLMVCGKLNDNRIIQEFILNSSAPLDTAVKLSRALNITAFKEKERAMDLLNAAKYSENMATELLTIASGSTNAGFILRAVDHRCTTILDCLIECEQKDVVAHPAVQKYLTEVWYGNLDWTTWKLVLLFFTFLACPPVWLVFSLPLKHRFNKIPIMKFMSYLASHIFLIVLFILTIVYPPLSPIYEGHMVPCWNEWLLLAWLSGMLVSELTHHGERAGLAWIRVFVLGFSAAAFFCHLLAFLFSDRDRLHCLFARNILLGMAMTLSFVQFLEFLTFHHLFGPWAIIIRDLIKDLTRFAVILALFHVAFTMQLSAVYQPVYPDDTATQSLNETETVHSESNIQDPIDITVLLFFSLFGLVEPGCLPSLNRTPQFTFVIVRFVFGVYLIVTLIVLINLLIAMMSDTYQRIQAQSDTEWKFGRATLIRDMTRKPGTPSPFNLFTNLFYYIKLLCKHKGKLCSANARNLMSEEDIDGFSDTRSIDLLAQTSLSWIRTPAKRTMQVAPEGKNLHVLRVLLFCIPVNVKGKFST mint 30AB66B9979F3E39 nompc-pef1 2 Peflin 4 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cdna library cdna library fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association mint nf nompc-pef1-1 2 TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Peflin 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f1b mint nompc-pef1-2 2 Peflin 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain physical association physical association f1b mint nompc-pef1-3 3 TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 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 Peflin 4 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f2b mint nompc-pef1-4 3 TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 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 Peflin 4 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f3a mint While ANK1-2 pulled down the Ca2+-binding mutant and all truncated forms of peflin in significantly (p < 0.01) smaller amounts than full-length wildtype peflin (Fig. 3), the interaction was almost abolished for &#61508;NHB and &#61508; lacking the NHB domain (< 10% of protein pulled down in comparison to wt peflin; p << 0.01). These results suggest that peflin has several interaction sites with the ANK repeats of TRPN1 and that the NHB domain and EF5, which mediates dimerization, are especially important for binding ANK repeats. The interaction of ANK1-2 with the Ca2+ binding mutant of peflin was also strongly reduced (31 ± 9% of protein pulled down in comparison to wt peflin; p < 0.01), suggesting that the ability to bind Ca2+ is also important for binding TRPN1 ANK repeats. nompc-pef1-5 3 Peflin 4 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain TRPN1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp 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 site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain physical association physical association f3b mint We did similar pulldown assays with the C-terminal tail of TRPN1. As shown in figure 3B, the C-terminal tail pulled down the Ca2+-binding mutant of peflin equally well as wt peflin and it pulled down &#61508;NHB even better than wt (p < 0.01). In contrast, the three truncations lacking one or several EF-hands were pulled down in significantly (p < 0.05) smaller amounts than wt peflin (Fig. 3B). This reduction was especially prominent for &#61508;EF5 and &#61508;EF3-5 (< 20% of protein pulled down in comparison to wt peflin; p << 0.01), suggesting that EF5 and the ability of peflin to dimerize is particularly important for its interaction with the C-terminal tail of TRPN1. These results suggest that different surfaces of peflin interact with the ANK repeats and the C-terminal tail. While the presence of the NHB domain is important for the interaction with ANK1-2, it is dispensable and may even hinder the interaction with the C-terminal tail. nompc-pef1-6 1 Peflin 4 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined TRPN1 5 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain colocalization colocalization mint When co-expressed with mCherry-TRPN1, GFP-peflin changed its distribution and no longer localized in a punctate pattern but in a reticular pattern, similar to TRPN1 (Fig. 4B). This mCherry- TRPN1-dependent distribution of GFP-peflin overlapped with the PDI staining (Supplemental Fig. S2C), suggesting that in the presence of TRPN1 GFP-peflin was associated with the ER. Thus, coexpression of TRPN1 and peflin revealed that both proteins tightly interacted in CL4 cells. When we co-expressed deletion mutants of TRPN1 that either truncated ANK1-6 (&#61508;ANK1-6) or the C-terminal tail (&#61508;C) or both (&#61508;ANK1-6/C), peflin was no longer redistributed and localized in its typical punctate pattern (Fig. 4C), showing that both ANK1-6 and the C-terminal domain are necessary for the tight interaction of peflin with TRPN1 in living cells. cells. Together these results suggest that ANK1-2 tightly interact with peflin but when fused to the TRPN1 backbone this interaction needs the support of the Cterminal tail. Similarly, the interaction of peflin with the C-terminus fused to the TRPN1 backbone needs the support of the N-terminal ANK repeats. In agreement, neither of the peflin fragments was redistributed by TRPN1 in CL4 cells, as expected from the loss of their interaction with ANK1-2 in pulldown assays (Fig. 6B, C). f4b f4c f4d f6 sf2c nompc-pef1-7 1 TRPN1 5 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell rfp tag rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Peflin 4 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f5 mint Therefore, we asked whether peflin interacts with the ANK repeats of TRPN1 also in CL4 cells and fused ANK1-2 to RFP. RFPANK1- 2 was diffusely distributed in the cytoplasm of CL4 cells (Fig. 5A). After co-expression of RFP-ANK1-2 with GFP-peflin, RFP-ANK1-2 adopted the staining pattern of GFP-peflin: in addition to the diffuse distribution, RFP fluorescence co-localized with the GFP fluorescence to large intracellular punctae. This result suggests that peflin interacts with the isolated two N-terminal ANK repeats of TRPN1 also in CL4 cells. Together these results suggest that ANK1-2 tightly interact with peflin but when fused to the TRPN1 backbone this interaction needs the support of the Cterminal tail.