A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers

Elizabeth A. Booth; Eleanor W. Vane; Dustin Dovala; Jeremy Thorner

doi:10.1074/jbc.M115.683128

A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers*

From the ^‡Division of Biochemistry, Biophysics, and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 and
the ^§Program in Microbial Pathogenesis and Host Defense, Department of Microbiology and Immunology, University of California School of Medicine, San Francisco, California 94158-2200

↵4 To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Univ. of California, Rm. 526, Barker Hall, Berkeley, CA 94720-3202. Tel.: 510-642-2558; Fax: 510-642-6420; E-mail: jthorner{at}berkeley.edu.

↵1 Present address: Grifols Diagnostic Solutions Inc., 4560 Horton St., Emeryville, CA 94608.
↵2 Present address: Dept. of Medicinal Chemistry, School of Pharmacy, Univ. of Washington, Seattle, WA 98195.
↵3 Present address: Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, CA 94608.

Background: Septins self-assemble into hetero-octameric rods and higher order structures and recruit other proteins.

Results: A spectroscopic method (FRET) to measure septin interactions and binding of associated proteins was developed.

Conclusion: End to end polymerization of Cdc11-capped rods, heterotypic end to end junctions between Cdc11-capped rods and Shs1-capped rods, and binding of an associated protein were demonstrated.

Significance: This spectroscopic assay provides new insights about these polymeric proteins.

Abstract

Prior studies in both budding yeast (Saccharomyces cerevisiae) and in human cells have established that septin protomers assemble into linear hetero-octameric rods with 2-fold rotational symmetry. In mitotically growing yeast cells, five septin subunits are expressed (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) and assemble into two types of rods that differ only in their terminal subunit: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. EM analysis has shown that, under low salt conditions, the Cdc11-capped rods polymerize end to end to form long paired filaments, whereas Shs1-capped rods form arcs, spirals, and rings. To develop a facile method to study septin polymerization in vitro, we exploited our previous work in which we generated septin complexes in which all endogenous cysteine (Cys) residues were eliminated by site-directed mutagenesis, except an introduced E294C mutation in Cdc11 in these experiments. Mixing samples of a preparation of such single-Cys containing Cdc11-capped rods that have been separately derivatized with organic dyes that serve as donor and acceptor, respectively, for FRET provided a spectroscopic method to monitor filament assembly mediated by Cdc11-Cdc11 interaction and to measure its affinity under specified conditions. Modifications of this same FRET scheme also allow us to assess whether Shs1-capped rods are capable of end to end association either with themselves or with Cdc11-capped rods. This FRET approach also was used to follow the binding to septin filaments of a septin-interacting protein, the type II myosin-binding protein Bni5.

Footnotes

↵* This work was supported by an Amgen Scholars Summer Research Fellowship at University of California Berkeley (to E. W. V.) and by National Institutes of Health R01 Grants GM21841 (to J. T.) and GM101314 (to J. T. and Berkeley colleague, Prof. Eva Nogales, jointly). The authors declare that they have no conflicts of interest with the contents of this article.
↵ This article contains supplemental Figs. S1–S4.