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A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers
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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653696/ https://www.ncbi.nlm.nih.gov/pubmed/26416886 http://dx.doi.org/10.1074/jbc.M115.683128 |
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author | Booth, Elizabeth A. Vane, Eleanor W. Dovala, Dustin Thorner, Jeremy |
author_facet | Booth, Elizabeth A. Vane, Eleanor W. Dovala, Dustin Thorner, Jeremy |
author_sort | Booth, Elizabeth A. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4653696 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-46536962015-11-24 A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers Booth, Elizabeth A. Vane, Eleanor W. Dovala, Dustin Thorner, Jeremy J Biol Chem Cell Biology 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. American Society for Biochemistry and Molecular Biology 2015-11-20 2015-09-28 /pmc/articles/PMC4653696/ /pubmed/26416886 http://dx.doi.org/10.1074/jbc.M115.683128 Text en © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) . |
spellingShingle | Cell Biology Booth, Elizabeth A. Vane, Eleanor W. Dovala, Dustin Thorner, Jeremy A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title | A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title_full | A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title_fullStr | A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title_full_unstemmed | A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title_short | A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers |
title_sort | förster resonance energy transfer (fret)-based system provides insight into the ordered assembly of yeast septin hetero-octamers |
topic | Cell Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653696/ https://www.ncbi.nlm.nih.gov/pubmed/26416886 http://dx.doi.org/10.1074/jbc.M115.683128 |
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