Abstract
Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.
Highlights
Septins form a class of eukaryotic guanine nucleotide-binding proteins that were first identified in the budding yeast, Saccharomyces cerevisiae, and congregate in a ring at the bud neck during cell division [1,2,3,4]
At least 10 mammalian septins have been identified to date, but most have not been studied in any detail. (Note that many have been given multiple different names, which can cause confusion in the literature and, for this reason, we will use a standard nomenclature in this report, in accordance with Human Genome Organization (HUGO) and Mouse Genomic Nomenclature (MGN) committee guidelines; see Ref. 14)
Expression of Recombinant Septin Monomers, Dimers, and Trimers in E. coli—A fundamental problem in studying septins has been the absence of systems for their expression as stable, soluble proteins in bacteria
Summary
Septins form a class of eukaryotic guanine nucleotide-binding proteins that were first identified in the budding yeast, Saccharomyces cerevisiae, and congregate in a ring at the bud neck during cell division [1,2,3,4]. The Drosophila filaments appear to be composed of three different septins Two of these septins, Pnut and Sep, have been localized to the cleavage furrow in dividing cells, and Pnut has been implicated in cytokinesis, only in certain cell types, and RNA interference of Pnut expression has an unusually low penetrance [11,12,13]. It is of interest that in budding yeast, which does not contain any Borg-like genes, Cdc has been implicated in the regulation of septin organization at the bud neck [22, 23]. The mechanism by which Borgs bind to and regulate septins is not understood and has been frustrated by the lack of information on septin oligomerization and filament assembly.
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