Dissecting the nucleotide binding properties of the septins from S. cerevisiae

Abstract

Septins are a conserved family of guanosine triphosphate (GTP)-binding proteins that assemble into an ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. They are present in all higher eukaryotes except plants. Septins belong structurally to the P-Loop nucleoside triphosphatase (NTPases) like Rab and Ras. However, unlike other small guanosine triphosphatase (GTPases) septins are supposed to act as scaffolds rather than signalling mediators. This is why they are considered as the fourth class of cytoskeletal proteins. It is assumed that septins fulfil their functions independently of the bound nucleotide. The role of guanosine diphosphosphate (GDP) and GTP binding and subsequent hydrolysis was controversial debated in the last couple of years. Lack of crystal structures of yeast septin subunits or rods and difficulties to isolate single monomeric septin subunits often hindered the correlation of results obtained from in vivo studies with biochemical data. Recently, nucleotide binding and hydrolysis was connected to the formation of septin rods from its subunits. However, the evidence was only indirectly obtained through the use of septin mutants in the context of intact cells. We provide here mechanistic insight into the nucleotide binding of the yeast septins by in vitro assays using purified septin rods and building blocks, thereby adding further insights to the already available models on septin filament formation.

Biochemical characterization of a human septin octamer

“…4). This is more in line with our findings for yeast septins (Baur et al 2018) rather than with the properties of small GTPases of the Ras family.…”

“…We have previously measured the uptake of [γ 32 P] labelled GTP in the presence of EDTA by the hexameric- and octameric septin rods from yeast (Baur et al 2018). We subjected our human septin rods to the same assay.…”

“…Nucleotide uptake-and hydrolysis assays Uptake of [γ 32 P]-GTP to hexameric and octameric rods and isolated SEPT9 was performed as described elsewhere for yeast septins (Baur et al 2018). Briefly, septin preparations were incubated with [γ 32 P]-GTP in exchange buffer containing 5 mM EDTA.…”

Biochemical Characterization of a Human Septin Octamer

“…This indicates that bound nucleotides and presumable hydrolysis products remain in the binding pocket and are not released into the surrounding medium, at least not in the timeframe of the experiment (120-180 min) and at least not under the conditions applied. This is more in line with our findings for yeast septins (Baur et al, 2018) rather than with the properties of small GTPases of the Ras family.…”

DataSheet1.pdf

“…Furthermore, in yeast, one of the catalytically inactive subunits (Cdc3) is bound to GTP, as anticipated, while the other (Cdc11) is hypothetically bound to GDP, presumably acquired from the cytosol (Farkasovsky et al, 2005;Weems and McMurray, 2017). However, another model claims that Cdc3 and Cdc11 are apoproteins even when incorporated into octamers (Baur et al, 2019). This conundrum is expected to be resolved once more structural information on yeast septins becomes available.…”