Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

DC FieldValueLanguage
dc.contributor.authorCabukusta, Birol
dc.contributor.authorKohlen, Jan A.
dc.contributor.authorRichter, Christian P.
dc.contributor.authorYou, Changjiang
dc.contributor.authorHolthuis, Joost C. M.
dc.date.accessioned2021-12-23T16:11:33Z-
dc.date.available2021-12-23T16:11:33Z-
dc.date.issued2016
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/9766-
dc.description.abstractSingle-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of He La cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.
dc.description.sponsorshipEuropean Union 7th FP Marie-Curie ITN ``Sphingonet'' Grant [289278]; Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [Sonderforschungsbereich/SFB944-P14]; This work was supported by the European Union 7th FP Marie-Curie ITN ``Sphingonet'' Grant 289278 and by Deutsche Forschungsgemeinschaft Sonderforschungsbereich/SFB944-P14 (to J. C. M. H.). The authors declare that they have no conflicts of interest with the contents of this article.
dc.language.isoen
dc.publisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
dc.relation.ispartofJOURNAL OF BIOLOGICAL CHEMISTRY
dc.subjectBiochemistry & Molecular Biology
dc.subjectCANCER CELLS
dc.subjectCURCUMIN
dc.subjectDYNAMICS
dc.subjectIDENTIFICATION
dc.subjectPHOSPHORYLATION
dc.subjectPROTEIN SMSR
dc.subjectRADIATION-INDUCED APOPTOSIS
dc.subjectSPHINGOLIPID BIOSYNTHESIS
dc.subjectSPHINGOMYELIN SYNTHASES
dc.subjectSTOICHIOMETRY
dc.titleMonitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching
dc.typejournal article
dc.identifier.doi10.1074/jbc.M116.749812
dc.identifier.isiISI:000388875500031
dc.description.volume291
dc.description.issue47
dc.description.startpage24735
dc.description.endpage24746
dc.contributor.orcid0000-0002-7839-6397
dc.contributor.orcid0000-0001-8912-1586
dc.contributor.researcheridL-3901-2014
dc.contributor.researcheridAAL-9157-2020
dc.identifier.eissn1083351X
dc.publisher.place9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
dcterms.isPartOf.abbreviationJ. Biol. Chem.
dcterms.oaStatusGreen Published, hybrid
crisitem.author.deptSonderforschungsbereich 944: Physiologie und Dynamik zellulärer Mikrokompartimente-
crisitem.author.deptidorganisation19-
crisitem.author.orcid0000-0002-7839-6397-
crisitem.author.parentorgFB 05 - Biologie/Chemie-
crisitem.author.grandparentorgUniversität Osnabrück-
crisitem.author.netidYoCh745-
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