title: Detection of protein-protein interactions at the septin collar in Saccharomyces cerevisiae using a tripartite split-GFP system.
abstract: Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck. We show, first, that for septin-septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a "molecular ruler." Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Finnigan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202.
authors.forename: Angela
authors.initials: A
authors.lastname: Duvalyan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202.
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Liao
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202.
authors.forename: Aspram
authors.initials: A
authors.lastname: Sargsyan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202 jthorner@berkeley.edu.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D004718:Saccharomycetales / Q000378:metabolism; D058112:Septins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: C100763:BUD4 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C116847:HOF1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; C464019:NIS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D058112:Septins