Septin Phosphorylation and Coiled-Coil Domains Function in Cell and Septin Ring Morphology in the Filamentous Fungus Ashbya gossypii

Abstract

Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function.

Septin Form and Function at the Cell Cortex

“…In addition, most septins are predicted to have a C-terminal coiled coil domain. This region remains functionally mysterious as it has failed to resolve by x-ray crystallography, possesses high flexibility, and produces variable phenotypes when removed from individual septins (28,29,33,34). These rod-shaped complexes serve as the basic subunit of filament assembly.…”

Forging the ring: from fungal septins' divergent roles in morphology, septation and virulence to factors contributing to their assembly into higher order structures

“…Phosphomimetics of all Shs1 sites (Shs1 9D) is recessive lethal. Shs1 9A has decreased mobility and Shs1 9D shows increase dynamics in interregion rings as measured by FRAP (Meseroll et al, 2013). Thus, ring dynamics could be regulated through phosphorylation of Shs1.…”

“…In fungi, septins are well-known for their participation in septation and spore production (Hartwell, 1971;Hernandez-Rodriguez et al, 2012;Lindsey et al, 2010a;Meseroll et al, 2012;Meseroll et al, 2013). In filamentous ascomycetes, deletion of septin genes does not abolish septation but significantly reduces the number of septa in the hyphae (Berepiki & Read, 2013;Chen et al, 2016;Dagdas et al, 2012;Hernandez-Rodriguez et al, 2012;Lindsey et al, 2010a;Vargas-Muñiz et al, 2015).…”

“…cerevisiae, Ash. gossypii and F. graminearum, septins are involved in ascospore formation (Chen et al, 2016;Fares et al, 1996;Heasley & McMurray, 2016;Meseroll et al, 2013). Similarly, in N. crassa, Asp.…”

“…gossypii, Cdc11, Cdc12, Cdc2 and Shs1 are also phosphorylated in vivo (Meseroll et al, 2013). Phosphorylation site mutations in Cdc11 (S314A) and Cdc3 (S91D) resulted in aberrant spore morphology, and mutated septins failed to localize to any structure.…”