| The septins are a conserved protein family involved in cytokinesis and other processes. In budding yeast, these proteins are thought to form a filament structure at the mother-bud neck, called the neck filaments. Given the potential importance of filament formation in septin function, we have developed a purified system to study the polymerization and structure of septins in budding yeast. We have shown that a complex containing the septins, Cdc3p, Cdc10p, Cdc11p, and Cdcl2p, formed long filaments and higher order structures in vitro. Complexes from cells deleted for CDC10 or CDC11 were not capable of polymerization. Analysis of the neck region by electron microscopy revealed that cdc10Delta and cdc11Delta cells did not contain neck filaments. These results strengthen the hypothesis that the neck filaments are comprised of septin polymers. Surprisingly, cytokinesis occurred in cdc10Delta cells, suggesting that septins, may be able to function in the absence of normal polymerization and formation of a higher order filament structure.; To test models for septin complex and filament organization, we purified septins from mutants predicted to affect neck filament structure, cdc3COIL and gin4Delta. We found that the lengthened coiled-coil domain of cdc3COIL had no discernable effect on septin complex architecture but did disrupt polymerization. cdc3COIL mutants with detectable staining at the neck were able to cytokinese, consistent with data correlating septin function with localization rather than filament assembly in cdc10Delta and gin4Delta cells. Septin-dependent processes are near normal in gin4Delta cells, though the septins are observed to undergo a dramatic reorganization from a ring to discrete bars running through the mother-bud neck (Longtine at al., 1998). Based on these observations, Longtine et al. proposed that Gin4p plays a central role in neck filament organization by linking septin filaments. We found that deleting GIN4 had a pronounced effect on septin complex stability and polymerization, suggesting that this kinase may play a more complex role in septin filament assembly and/or organization than described by this model. |
