Biochemical Characterization of a Human Septin Octamer.

Martin Fischer,Thomas Gronemeyer,Reinhild Rösler,Nils Johnsson,Dominik Frank

doi:10.3389/fcell.2022.771388

Martin Fischer, Thomas Gronemeyer + Show 3 more

Open Access

https://doi.org/10.3389/fcell.2022.771388

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Abstract

Septins are part of the cytoskeleton and polymerize into non-polar filaments of heteromeric hexamers or octamers. They belong to the class of P-loop GTPases but the roles of GTP binding and hydrolysis on filament formation and dynamics are not well understood. The basic human septin building block is the septin rod, a hetero-octamer composed of SEPT2, SEPT6, SEPT7, and SEPT9 with a stoichiometry of 2:2:2:2 (2-6-7-9-9-7-6-2). Septin rods polymerize by end-to-end and lateral joining into linear filaments and higher ordered structures such as rings, sheets, and gauzes. We purified a recombinant human septin octamer from E. coli for in vitro experimentation that is able to polymerize into filaments. We could show that the C-terminal region of the central SEPT9 subunit contributes to filament formation and that the human septin rod decreases the rate of in vitro actin polymerization. We provide further first kinetic data on the nucleotide uptake- and exchange properties of human hexameric and octameric septin rods. We could show that nucleotide uptake prior to hydrolysis is a dynamic process and that a bound nucleotide is exchangeable. However, the hydrolyzed γ-phosphate is not released from the native protein complex. We consequently propose that GTP hydrolysis in human septins does not follow the typical mechanism known from other small GTPases.

Highlights

Septins were discovered in yeast in 1971 (Hartwell 1971) and are part of the cytoskeleton (Mostowy and Cossart 2012)
SEPT6 and SEPT7 were expressed from one plasmid and SEPT2 from the second plasmid as in a previous study (Sheffield et al, 2003)
Complexes eluted in a shoulder in the second half of the main peak and in a minor peak directly adjacent to the main peak (Supplementary Figure S1A)

Summary

Introduction

Septins were discovered in yeast in 1971 (Hartwell 1971) and are part of the cytoskeleton (Mostowy and Cossart 2012). They form non-polar filaments that unlike actin filaments and microtubules do not serve as railroad tracks for motor proteins. Being rather neglected for several years after their discovery, research on the septins gained a growing attention over the past few years (Menon and Gaestel, 2017). Labeled as passive scaffold proteins, septins were later discovered to actively participate in many dynamic intracellular processes. The septin subunit SEPT9 was reported to bundle actin and microtubules (Bai et al, 2013; Dolat et al, 2014; Mavrakis et al, 2014; Smith et al, 2015) Septins regulate the organization of the cytoskeleton, vesicle transport and fusion, chromosome alignment and segregation, and cytokinesis (Surka et al, 2002; Estey et al, 2010; Bowen et al, 2011; Füchtbauer et al, 2011; Tokhtaeva et al, 2015).

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