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 Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. FEBS Lett. (0014-5793) 2013 Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. ecoli Escherichia coli (strain K12) crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. FEBS Lett. (0014-5793) 2013 Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. FEBS Lett. (0014-5793) 2013 Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. in vitro In vitro competition binding competition binding western blot western blot Immuno blot Immuno blot Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. FEBS Lett. (0014-5793) 2013 Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex. FEBS Lett. (0014-5793) 2013 Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Mizutani M., Mukaiyama K., Xiao J., Mori M., Satou R., Narita S., Okuda S., Tokuda H. FEBS Lett. (0014-5793) 2013 lola_ecoli Outer-membrane lipoprotein carrier protein yzzV P20 lolA lplA b0891 JW0874 protein protein ecoli Escherichia coli (strain K12) MKKIAITCALLSSLVASSVWADAASDLKSRLDKVSSFHASFTQKVTDGSGAAVQEGQGDLWVKRPNLFNWHMTQPDESILVSDGKTLWFYNPFVEQATATWLKDATGNTPFMLIARNQSSDWQQYNIKQNGDDFVLTPKASNGNLKQFTINVGRDGTIHQFSAVEQDDQRSSYQLKSQQNGAVDAAKFTFTPPQGVTVDDQRK DIP protein P61316 original sequence version: 77 F2884D82D8DFEF1D lole_ecoli Lipoprotein-releasing system transmembrane protein LolE lolE b1118 JW1104 ycfW protein protein ecoli Escherichia coli (strain K12) MAMPLSLLIGLRFSRGRRRGGMVSLISVISTIGIALGVAVLIVGLSAMNGFERELNNRILAVVPHGEIEAVDQPWTNWQEALDHVQKVPGIAAAAPYINFTGLVESGANLRAIQVKGVNPQQEQRLSALPSFVQGDAWRNFKAGEQQIIIGKGVADALKVKQGDWVSIMIPNSNPEHKLMQPKRVRLHVAGILQLSGQLDHSFAMIPLADAQQYLDMGSSVSGIALKMTDVFNANKLVRDAGEVTNSYVYIKSWIGTYGYMYRDIQMIRAIMYLAMVLVIGVACFNIVSTLVMAVKDKSGDIAVLRTLGAKDGLIRAIFVWYGLLAGLFGSLCGVIIGVVVSLQLTPIIEWIEKLIGHQFLSSDIYFIDFLPSELHWLDVFYVLVTALLLSLLASWYPARRASNIDPARVLSGQ 37B73A6E5DC58517 pal_ecoli Peptidoglycan-associated lipoprotein pal excC b0741 JW0731 Tol-Pal system lipoprotein Pal protein protein ecoli Escherichia coli (strain K12) MQLNKVLKGLMIALPVMAIAACSSNKNASNDGSEGMLGAGTGMDANGGNGNMSSEEQARLQMQQLQQNNIVYFDLDKYDIRSDFAQMLDAHANFLRSNPSYKVTVEGHADERGTPEYNISLGERRANAVKMYLQGKGVSADQISIVSYGKEKPAVLGHDEAAYSKNRRAVLVY DIP protein P0A912 original sequence version: 52 449F9959C0274430 lole-pal 3 LolE 5 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified Pal 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction mint On the other hand, Pal with pBPA at positions 4 and 5 generated crosslinked products that migrated to ~70 kDa only with LolE (Fig. 2A and B). The LolCDE complex liganded with Pal containing pBPA at position 4 was subjected to photo-crosslinking after incubation with various nucleotides (Fig. 3). Addition of ATP significantly decreased the amount of the LolE-Pal crosslinked product whereas neither ADP nor AMP decreased the crosslinking (Fig. 3A and B). f2a f2b f3 f4a pal-1 3 Pal 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Stoichiometry: 2.0 direct interaction direct interaction f2b mint The Pal dimer was also detected in the isolated liganded LolCDE preparation, suggesting that affinity purification with TALON resin also purified free Pal possessing a His-tag at its C-terminus. When Pal contained pBPA at position 5, two other crosslinked products were generated (Fig. 2B). lole-pal-1 1 LolE 5 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell Pal 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f1a f1c mint Therefore, Pal containing pBPA was overexpressed with or without co-overexpression of an interaction partner, LolCDE. The cells were harvested and then subjected to UV irradiation, followed by SDS-PAGE and immunoblotting with anti-LolC, -LolD, -LolE and -Pal antibodies (Fig. 1). Overexpressed Pal reacted with not only anti-Pal but also anti-LolE antibodies and migrated near the front of the gel (Fig. 1A and C). lola-pal 2 Pal 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined LolA 4 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified physical association physical association mint LolCDE liganded with Pal containing pBPA at position 4 was incubated with or without ATP and/or LolA and then subjected to crosslinking (Fig. 4). Addition of LolA in the absence of ATP only marginally affected the level of LolE-Pal crosslinking. When ATP and LolA were added together, LolE-Pal crosslinking significantly decreased and was almost undetectable with anti-Pal antibodies. Instead, the LolA-Pal crosslinked product was prominently generated in the presence of both ATP and LolA (Fig. 4C). The effects of ATP binding and hydrolysis on the generation of LolE-Pal and LolA-Pal crosslinking were further examined (Fig. 5). Both AMP-PNP added at 20 mM and ATP added in the absence of Mg2+ decreased the amount of crosslinked LolE-Pal to some extent but did not cause generation of the LolA-Pal crosslinked product (Fig. 5C). On the other hand, addition of ATP in the presence of Mg2+ nearly completely abolished the crosslinking between LolE and Pal, but caused crosslinking between LolA and Pal, indicating that ATP hydrolysis is required for the transfer of Pal from LolE to LolA. f4c f5