Biochemical Characterization of a Human Septin Octamer

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.

Keywords: GTPases; actin polymerization; nucleotide uptake; septin octamer; septins.

FIGURE 1

Purification of octameric septin rods. (A) SDS-PAGE (Coomassie staining) of a representative purification of septin octamers containing SEPT9_FL and SEPT9_G568. Fractions from IMAC and SEC including the major and minor peak are indicated. SEPT9 degradation products are marked with arrows. The anti-SEPT9 Western blot shows the SEPT9 degradation products. (B) Representative MS analysis of the purified septin complexes. Both the main product peak and the minor peak from SEC were analyzed. Peptide abundancies of the SEPT_FL (left panel) and SEPT9_G568 (right panel) octamer are plotted; the indicated intensity score reflects the accumulated intensities of all peptides detected for the respective subunit. (C) Nucleotide content of octameric rods containing SEPT9_FL or SEPT9_G568 detected by analytical anion exchange chromatography (representative runs out of at least three per rod species performed) after heat denaturation of the protein. Calibration runs with GTP and GDP are shown in the upper row.

FIGURE 2

Electron microscopy of SEPT9_FL and SEPT9_G568 containing septin rods. Negative stains of septin filaments obtained by dialysis in low salt buffer showing bundles and network-like structures. (A) Electron microscopy of septin filaments containing SEPT9_FL. (B) Electron microscopy of septin filaments containing SEPT9_G568.

FIGURE 3

Influence of human septin rods on actin polymerization. (A) Actin polymerization assay using pyrene actin with and without septin hexamers. (B) Actin polymerization assay using pyrene actin with and without SEPT9_G568 containing octamers. The assays shown were performed in triplicate and in quintuplicate for actin. The same averaged actin curve was used for evaluation of the assay. The unprocessed raw data are shown in Supplementary Figure S4.

FIGURE 4

Nucleotide uptake- and hydrolysis properties of hexameric and SEPT9_G568 octameric septin rods. (A) Nucleotide exchange reaction of hexameric (left panel) and octameric septins (right panel). Purified complexes were incubated with [γ³²P]-GTP and uptake was monitored at the indicated timepoints by a filter assay. [CPM%] values (normalized radioactive counts) are plotted vs. the reaction time. The connecting line represents the fitting curve of the exponential association. (B) Nucleotide exchange reaction of octameric septins preloaded with [γ³²P]-GTP. The preloaded complex was incubated with GTP and decrease of [γ³²P]-GTP was monitored at the indicated timepoints by a filter assay as in A. The connecting line represents the fitting curve of the exponential dissociation. (C) Compilation of the t_1/2 values of all performed nucleotide exchange assays. The parameters of the statistical evaluation are provided in Supplementary Tables S2, S3. Error bars represent the standard deviation. All assays [including those depicted in detail in (A,B)] were performed at least in triplicate. (D) Representative [γ³²P]-GTP uptake assay for isolated SEPT9_FL (left panel) and SEPT9_Q295-E567 (right panel) subunits. The assay was performed as in (A). The raw, unprocessed CPM counts are plotted vs. the reaction time. For comparison, a representative nucleotide uptake reaction for a septin octamer is also plotted. The inlets show the Coomassie stained SDS-PAGE of both purifications. (E) Representative GTP hydrolysis assay for octameric septins. After preloading with [γ³²P]-GTP, the complex was subjected to hydrolysis conditions applicable for small GTPases. The reaction was performed at 25°C (left panel) and 37°C (right panel). Samples were taken at the indicated time points and the [γ³²P]-GTP content was detected by a filter assay. [CPM%] values (normalized radioactive counts) are plotted vs. the reaction time. GTP hydroloysis of hexameric rods is shown in Supplementary Figure S4.