Decision letter: Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

ABSTRACTSeptin proteins co-assemble into hetero-oligomers that polymerize into cytoskeletal filaments with a variety of cellular functions. In Saccharomyces cerevisiae, where septins were first discovered, five subunits comprise two species of septin hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10– Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Septins evolved from ancestral GTPases. We previously found evidence that slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1 into septin complexes. It was unclear why many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that the small molecule guanidine hydrochloride (GdnHCl) rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3– Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium ion (Gdm) occupies the site of a “missing” Arg sidechain that is present in other fungal species in which (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers co-exist with octamers in wild-type cells. These findings support a model in which Gdm reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to hetero-octamers. Given that septin hexamers made natively in human cells also exclude Cdc10-like central subunits via homodimerization of an active GTPase, our results provide new mechanistic details that likely apply to septin assembly throughout phylogeny.

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Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

eLife ◽

10.7554/elife.54355 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Courtney R Johnson ◽

Marc G Steingesser ◽

Andrew D Weems ◽

Anum Khan ◽

Amy Gladfelter ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Budding Yeast ◽

Guanidine Hydrochloride ◽

Fungal Species ◽

Side Chain ◽

Gtpase Activity ◽

Fungal Evolution ◽

Assembly Pathway

Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3–Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium occupies the site of a ‘missing’ Arg side chain found in other fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers natively co-exist with octamers. We propose that guanidinium reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to octamers. Since homodimerization by a GTPase-active human septin also creates hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply throughout phylogeny.

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The step-wise pathway of septin hetero-octamer assembly in budding yeast

eLife ◽

10.7554/elife.23689 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 26

Author(s):

Andrew Weems ◽

Michael McMurray

Keyword(s):

Budding Yeast ◽

Underlying Mechanism ◽

Guanine Nucleotides ◽

Gtp Hydrolysis ◽

The Core ◽

Assembly Pathway ◽

N Terminus ◽

Specific Affinity ◽

Stepwise Assembly

Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3–Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11–Cdc12–Cdc3 or Shs1–Cdc12–Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.

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Hsp104-dependent ability to assimilate mannitol and sorbitol conferred by a truncated Cyc8 with a C-terminal polyglutamine in Saccharomyces cerevisiae

PLoS ONE ◽

10.1371/journal.pone.0242054 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0242054

Author(s):

Hideki Tanaka ◽

Kousaku Murata ◽

Wataru Hashimoto ◽

Shigeyuki Kawai

Keyword(s):

Saccharomyces Cerevisiae ◽

Budding Yeast ◽

Spontaneous Mutation ◽

Guanidine Hydrochloride ◽

Transcriptional Corepressor ◽

Nonsense Mutations

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature.

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On the Preparation of Bovine α-Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646669 ◽

1978 ◽

Vol 39 (01) ◽

pp. 193-200 ◽

Cited By ~ 7

Author(s):

Erwin F Workman ◽

Roger L Lundblad

Keyword(s):

Immobilized Enzyme ◽

Catalytic Properties ◽

Specific Activity ◽

Guanidine Hydrochloride ◽

Low Molecular Weight ◽

Reversible Process ◽

Gel Beads ◽

Tissue Thromboplastin ◽

C 50 ◽

Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

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