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 Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus. Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus. FEBS letters 2016 imex curation Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk 11-Apr-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk Accepted 2016-MAR-30 AT 10:55 BST AT 10:55 BST by MARTA FEBS letters 2016 Only protein-protein interactions imex curation imex curation Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus. Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus. FEBS letters 2016 imex curation Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk 11-Apr-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk Accepted 2016-MAR-30 AT 10:55 BST AT 10:55 BST by MARTA FEBS letters 2016 Only protein-protein interactions imex curation imex curation essc_staae Type VII secretion system protein EssC essC NWMN_0223 protein protein staae Staphylococcus aureus (strain Newman) MHKLIIKYNKQLKMLNLRDGKTYTISEDERADITLKSLGEVIHLEQNNQGTWQANHTSINKVLVRKGDLDDITLQLYTEADYASFAYPSIQDTMTIGPNAYDDMVIQSLMNAIIIKDFQSIQESQYVRIVHDKNTDVYINYELQEQLTNKAYIGDHIYVEGIWLEVQADGLNVLSQNTVASSLIRLTQEMPHAQADDYNTYHRSPRIIHREPTDDIKIERPPQPIQKNNTVIWRSIIPPLVMIALTVVIFLVRPIGIYILMMIGMSTVTIVFGITTYFSEKKKYNKDVEKREKDYKAYLDNKSKEINKAIKAQRFSLNYHYPTVAEIKDIVETKAPRIYEKTSHHHDFLHYKLGIANVEKSFKLDYQEEEFNQRRDELFDDAKELYEFYTDVEQAPLINDLNHGPIAYIGARHLILEELEKMLIQLSTFHSYHDLEFLFVTREDEVETLKWARWLPHMTLRGQNIRGFVYNQRTRDQILTSIYSMIKERIQAVRERSRSNEQIIFTPQLVFVITDMSLIIDHVILEYVNQDLSEYGISLIFVEDVIESLPEHVDTIIDIKSRTEGELITKEKELVQLKFTPENIDNVDKEYIARRLANLIHVEHLKNAIPDSITFLEMYNVKEVDQLDVVNRWRQNETYKTMAVPLGVRGKDDILSLNLHEKAHGPHGLVAGTTGSGKSEIIQSYILSLAINFHPHEVAFLLIDYKGGGMANLFKDLVHLVGTITNLDGDEAMRALTSIKAELRKRQRLFGEHDVNHINQYHKLFKEGIATEPMPHLFIISDEFAELKSEQPDFMKELVSTARIGRSLGIHLILATQKPSGVVDDQIWSNSKFKLALKVQDRQDSNEILKTPDAADITLPGRAYLQVGNNEIYELFQSAWSGATYDIEGDKLEVEDKTIYMINDYGQLQAINKDLSGLEDEETKENQTELEAVIDHIESITTRLEIEEVKRPWLPPLPENVYQEDLVETDFRKLWSDDAKEVELTLGLKDVPEEQYQGPMVLQLKKAGHIALIGSPGYGRTTFLHNIIFDVARHHRPDQAHMYLFDFGTNGLMPVTDIPHVADYFTVDQEDKIAKAIRIFNDEIDRRKKILSQYRVTSISEYRKLTGETIPHVFILIDNFDAVKDSPFQEVFENMMIKMTREGLALDMQVTLTASRANAMKTPMYINMKTRIAMFLYDKSEVSNVVGQQKFAVKDVVGRALLSSDDNVSFHIGQPFKHDETKSYNDQINDEVSAMTEFYKGETPNDIPMMPDEIKYEDYRESLNLPDIVANGALPIGLDYEGVTLQKIKLTEPAMISSENPREIAHIAEIMMKEIDILNEKYAICIADSSGEFKAYRHQVANFAEEREDIKAIHQLMIEDLKQREMDGPFEKDSLYIINDFKTFIDCTYIPEDDVKKLITKGPELGLNILFVGIHKELIDAYDKQIDVARKMINQFSIGIRISDQQFFKFRFIQREPVIKENEAYMVANQAYQKIRWFK 0EC13F663848BBCB esaa_staae Type VII secretion system accessory factor EsaA esaA NWMN_0220 protein protein staae Staphylococcus aureus (strain Newman) MKKKNWIYALIVTLIIIIAIVSMIFFVQTKYGDQSEKGSQSVSNKNNKIHIAIVNEDQPTTYNGKKVELGQAFIKRLANEKNYKFETVTRNVAESGLKNGGYQVMIVIPENFSKLAMQLDAKTPSKISLQYKTAVGQKEEVAKNTEKVVSNVLNDFNKNLVEIYLTSIIDNLHNAQKNVGAIMTREHGVNSKFSNYLLNPINDFPELFTDTLVNSISANKDITKWFQTYNKSLLSANSDTFRVNTDYNVSTLIEKQNSLFDEHNTAMDKMLQDYKSQKDSVELDNYINALKQMDSQIDQQSSMQDTGKEEYKQTVKENLDKLREIIQSQESPFSKGMIEDYRKQLTESLQDELANNKDLQDALNSIKMNNAQFAENLEKQLHDDIVKEPDTDTTFIYNMSKQDFIAAGLNEGEANKYEAIVKEAKRYKNEYNLKKPLAEHINLTDYDNQVAQDTSSLINDGVKVQRTETIKSNDINQLTVATDPHFNFEGDIKINGKKYDIKDQSVQLDTSNKEYKVEVNGVAKLKKDAEKDFLKDKTMHLQLLFGQANRQDEPNDKKATSVVDVTLNHNLDGRLSKDALSQQLSALSRFDAHYKMYTDTKGREDKPFDNKRLIDMMVDQVINDMESFKDDKVAVLHQIDSMEENSDKLIDDILNNKKNTTKNKEDISKLIDQLENVKKTFAEEPQEPKIDKGKNDEFNTMSSNLDKEISRISEKSTQLLSDTQESKSIADSVSGQLNQVDNNVNKLHATGRALGVRANDLNRQMAKNDKDNELFAKEFKKVLQNSKDGDRQNQALKAFMSNPVQKKNLENVLANNGNTDVISPTLFVLLMYLLSMITAYIFYSYERAKGQMNFIKDDYSSKNHLWNNVITSGVIGTTGLVEGLIVGLIAMNKFHVLAGYRAKFILMVILTMMVFVLINTYLLRQVKSIGMFLMIAALGLYFVAMNNLKAAGQGVTNKISPLSYIDNMFFNYLNAEHPIGLVLVILTVLVIIGFVLNMFIKHFKKERLI F9E24F016B19F276 essb_staae Type VII secretion system protein EssB NWMN_0222 essB protein protein staae Staphylococcus aureus (strain Newman) MVKNHNPKNEMQDMLTPLDAEEAAKTKLRLDMREIPKSSIKPEHFHLMYLLEQHSPYFIDAELTELRDSFQIHYDINDNHTPFDNIKSFTKNEKLRYLLNIKNLEEVNRTRYTFVLAPDELFFTRDGLPIAKTRGLQNVVDPLPVSEAEFLTRYKALVICAFNEKQSFDALVEGNLELHKGTPFETKVIEAATLDLLTAFLDEQYQKQEQDYSQNYAYVRKVGHTVFKWVAIGMTTLSVLLIAFLAFLYFSVMKHNERIEKGYQAFVKDDYTQVLNTYDDLDGKKLDKEALYIYAKSYIQTNKQGLEKDKKENLLNNVTPNSNKDYLLYWMELGQGHLDEAINIATYLDDNDITKLALINKLNEIKNNGDLSNDKRSEETKKYNDKLQDILDKEKQVKDEKAKSEEEKAKAKDEKLKQQEENEKKQKEQAQKDKEKRQEAERKK FACCFE3353DE1F77 essa-1 2 5 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level In vivo crosslinking analysis of EssB resulted in no detectable EssB crosslinks unless the protein was overproduced, in which case a very faint crosslinked product running at the approximate size of an EssB homo-dimer could be detected (Fig 3B). As homo-dimerisation of EssB has been reported previously, [15, 16] it is likely that this crosslink is a homo-dimeric form of EssB. direct interaction direct interaction f2 f3 essa-2 2 4 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction f3 As shown in Fig. 3A, incubation with formaldehyde resulted in EsaA-specific crosslinks in whole cells of the wild-type strain. A series of 4–5 discrete bands could be detected, migrating around 250 kDa. Interestingly, essentially the same pattern of EsaA crosslinks was seen whether EssA, EssB or EssC were absent, suggesting that the crosslinked products did not contain any of these proteins. It has been reported that the long extracellular domain of YueB, the Bacillus subtilis homologue of EsaA, forms a highly elongated homo-dimer [17] and it is therefore likely that the crosslinks seen here correspond to different conformers of a crosslinked EsaA dimer. essc-1 2 3 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level 3 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction f3 Crosslinking of EssC in vivo also yielded high molecular weight products, as was observed in vitro (Fig 3C). However, in contrast to the in vitro analysis, where one major crosslinked product was seen, in this instance at least two distinct high molecular weight species were detected, both of which migrated above the 460 kDa molecular weight marker. The differences between the pattern of crosslinks seen in vivo and in vitro may reflect the difference in size between DSS which was used as a crosslinker in vitro and formaldehyde which was used in vivo. Alternatively they may arise due to the presence of ATP, proton-motive force or other factors that are present in whole cells. It should be noted that as similar crosslinks were seen in the absence of any of EsaA, EssA or EssB, neither of the two crosslinks arise from interactions of EssC with any of the other core membrane components of the Ess system, and probably represent homo-multimeric forms of EssC. essa-3 1 5 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction f5 However, bands containing EsaA and EssB were observed. As shown in Fig. 5(A), two complexes containing EsaA could be detected, one of which close to the 150 kDa marker and the second just above the 200 kDa marker, which could conceivably correspond to monomers and dimers of EsaA. In contrast, a single EssB-containing band was detected, migrating between the 66 kDa and 150 kDa marker, close to the expected size for an EssB dimer (Fig. 5B). Thus, given the differences in the masses of these bands these two proteins are highly unlikely to be part of the same complex. We confirmed this by repeating the BN PAGE on Fos-choline-12 membrane extracts from strains deleted for esaA or essB, and detected the same two EsaA complexes in the absence of EssB, and the same EssB complex in the absence of EsaA. We conclude that these two proteins do not interact with each other in isolated membranes. esaa-1 1 4 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction f5 However, bands containing EsaA and EssB were observed. As shown in Fig. 5(A), two complexes containing EsaA could be detected, one of which close to the 150 kDa marker and the second just above the 200 kDa marker, which could conceivably correspond to monomers and dimers of EsaA. In contrast, a single EssB-containing band was detected, migrating between the 66 kDa and 150 kDa marker, close to the expected size for an EssB dimer (Fig. 5B). Thus, given the differences in the masses of these bands these two proteins are highly unlikely to be part of the same complex. We confirmed this by repeating the BN PAGE on Fos-choline-12 membrane extracts from strains deleted for esaA or essB, and detected the same two EsaA complexes in the absence of EssB, and the same EssB complex in the absence of EsaA. We conclude that these two proteins do not interact with each other in isolated membranes.