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

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

doi:10.1074/jbc.m115.683128

Cited by 36 publications

(78 citation statements)

References 78 publications

(168 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Because the septin rod is ~32 nm in length, whereas each subunit itself has a diameter of ~4 nm (Fig. 1A), given the R 0 for the AF555–AF647 pair, FRET should be able to discriminate with adequate resolution interactions between septin subunits situated at different positions within the rod, or the interaction of a septin-interacting protein with septin subunits situated at different positions along the rod, as we have indeed demonstrated is the case (Booth et al, 2015). …”

Section: Protein Preparation and Labelingmentioning

confidence: 63%

“…To enable our FRET approach, into each Cys-less subunit, standard site-directed mutagenesis techniques were used to install a single Cys in place of the residue that, on the basis of sequence alignments and modeling (McMurray & Thorner, 2008) against the crystal structure of the human septin complex (Sirajuddin et al, 2007), should occupy a solvent-exposed position at the base of the predicted C-terminal extension (CTE) in Cdc11, Cdc12, Cdc3, and Shs1, and near the carboxyl terminus of Cdc10 (which is the only mitotic septin that lacks a CTE), yielding Cdc11(E294C), Cdc12(L310C), Cdc3(S407C), Shs1(E344C), and Cdc10(R298C) (Booth et al, 2015; de Val et al, 2013). By placing a single Cys at this location, it should be sufficiently accessible for efficient dye labeling, and the resulting conjugated fluorophore would be close to the globular GTP-binding domain and relatively free from constraints on its rotation.…”

Section: Protein Preparation and Labelingmentioning

confidence: 99%

“…We have established a FRET-based method for monitoring septin–septin interaction and demonstrated its importance and utility as a method for studying sep-tin assembly (Booth, Vane, Dovala, & Thorner, 2015). For example, in the case of Cdc11-capped mitotic heterooctamers, we anticipated that a batch of purified rods (Fig.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

Booth

Thorner

2016

Methods in Cell Biology

View full text Add to dashboard Cite

Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein–protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level “ruler” because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤ 10 nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins.

show abstract

Section: Protein Preparation and Labelingmentioning

confidence: 63%

Section: Protein Preparation and Labelingmentioning

confidence: 99%

See 1 more Smart Citation

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

Booth

Thorner

2016

Methods in Cell Biology

View full text Add to dashboard Cite

show abstract

“…Given this fact, the monitored energy transfer is an average dominated by the closest tags. We chose to use Alexa Fluor 488 as FRET donor and Alexa Fluor 555 as FRET acceptor, a conjugate couple largely used (29,30,31). FRET between this fluorophore pair is efficient in this context since the distance for 50% FRET (Förster distance, R o ) is 7.0 nm, compatible with the range 2.7 to 7.0 nm indicated above.…”

Section: Labeling Of H and L Ferritin Homopolymers For Fret Analysismentioning

confidence: 99%

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Carmona

Poli

Bertuzzi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

Background: The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H-and Ltype that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. Methods: We used the Fluorescence Resonance Energy Transfer (FRET) by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. Results: The FRET-efficiency increase from <0.3 of the disassembled to >0.7 in the reassembled allowed to study the self-assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. Conclusion: Heterodimeric (H/L) subunit association is preferred during H/L heteropolymers formation. The H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize and the ferroxidase activity of the heteropolymer reaches a plateau. General significance: This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Keywords

show abstract

“…Interactions between, for example, Cdc12-Cdc3, Cdc10-Cdc10 and Cdc11-Cdc11 at the so-called 'NC' interface are essential (Beise and Trimble, 2011;Sirajuddin et al, 2007). The terminal subunit Cdc11 can be replaced by an alternative septin subunit Shs1, resulting in the formation of alternative heterooctamers (Booth et al, 2015;Finnigan et al, 2015;Garcia et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Endosomal assembly and transport of heteromeric septin complexes promote septin cytoskeleton formation

Zander

Baumann

Weidtkamp‐Peters

et al. 2016

Journal of Cell Science

View full text Add to dashboard Cite

Septins are conserved cytoskeletal structures functioning in a variety of biological processes including cytokinesis and cell polarity. A wealth of information exists on the heterooligomeric architecture of septins and their subcellular localization at distinct sites. However, the precise mechanisms of their subcellular assembly and their intracellular transport are unknown. Here, we demonstrate that endosomal transport of septins along microtubules is crucial for formation of higher-order structures in the fungus Ustilago maydis. Importantly, endosomal septin transport is dependent on each individual septin providing strong evidence that septin heteromeric complexes are assembled on endosomes. Furthermore, endosomal trafficking of all four septin mRNAs is required for endosomal localization of their translation products. Based on these results, we propose that local translation promotes the assembly of newly synthesized septins in heteromeric structures on the surface of endosomes. This is important for the long-distance transport of septins and the efficient formation of the septin cytoskeleton.

show abstract

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

Cited by 36 publications

References 78 publications

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Endosomal assembly and transport of heteromeric septin complexes promote septin cytoskeleton formation

Contact Info

Product

Resources

About