Characterization of Septin Protein Interactions at the Yeast Bud Neck Using a Tripartite Split GFP Detection System (original) (raw)

The septin proteins are a conserved cytoskeletal element found across eukaryotes (including humans) that assemble into filamentous structures in vivo to (i) serve as a barrier between membrane-enclosed compartments, (ii) assist in promoting membrane curvature, and (iii) recruit and bind many non-septin protein targets [1-2]. In Saccharomyces cerevisiae there are five mitotically-expressed septins that are organized into the linear arrangement ("octamer") of Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 (with the terminal Cdc11 replaced with an alternate subunit, Shs1) [3]. Previous reports have suggested that nearly 100 other proteins are localized to the division site (bud neck) and may interact with the septins [2]. However, it remains unknown what contingent of proteins physically interacts with the septin structure, and if so, which position(s) along the length of the octamer are occupied. Previously, we demonstrated the use of a tripartite split GFP system to detect protein-protein interactions between nearby septin subunits and four non-septin binding partners [4-5]. Here, we have screened 25 additional candidates using this method by fluorescence microscopy in live cells.