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.

The taxonomy of the Mediterranean Aristolochia pallida complex has been under debate since several decades with the following species currently recognized: A . pallida , A . lutea , A . nardiana , A . microstoma , A . merxmuelleri , A . croatica , and A . castellana . These taxa are distributed from Iberia to Turkey. To reconstruct phylogenetic and biogeographic patterns, we employed cpDNA sequence variation using both noncoding (intron and spacer) and protein‐coding regions (i.e., trnK intron, matK gene, and trnK ‐ psbA spacer). Our results show that the morphology‐based traditional taxonomy was not corroborated by our phylogenetic analyses. Aristolochia pallida , A . lutea , A . nardiana , and A . microstoma were not monophyletic. Instead, strong geographic signals were detected. Two major clades, one exclusively occurring in Greece and a second one of pan‐Mediterranean distribution, were found. Several subclades distributed in Greece, NW Turkey, Italy, as well as amphi‐Adriatic subclades, and a subgroup of southern France and Spain, were revealed. The distribution areas of these groups are in close vicinity to hypothesized glacial refugia areas in the Mediterranean. According to molecular clock analyses the diversification of this complex started around 3–3.3 my, before the onset of glaciation cycles, and the further evolution of and within major lineages falls into the Pleistocene. Based on these data, we conclude that the Aristolochia pallida alliance survived in different Mediterranean refugia rarely with low, but often with a high potential for range extension, and a high degree of morphological diversity.

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 (2-7-6-9-9-6-7-2). 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 gamma-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.

No abstract