Detection of protein–protein interactions at the septin collar inSaccharomyces cerevisiaeusing a tripartite split-GFP system

Abstract

A tripartite split-GFP system faithfully reports the order of the subunits in septin hetero-octamers (and thus can serve as a “molecular ruler”), conversely yields little or no false signal even with very highly expressed cytosolic proteins, and detects authentic interactions of other cellular proteins that are bona fide septin-binding proteins.

Meeting report – shining light on septins

“…The budding yeast remains today a useful system to understand the role of septins and their cellular organization. Jeremy Thorner (University of California, Berkeley, USA) presented a tripartite split-GFP method as a means to interrogate the interactome of the filamentous septin collar (Finnigan et al, 2016). During the cell cycle, septins first form a patch at the presumptive bud site.…”

Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

“…Binding of Hsl1 to septins occurs via uniquely evolved septin-binding sequences (residues 611–950) downstream of its N-terminal kinase domain, which, to operate efficiently, must act in concert with a C-terminal phosphatidylserine-binding element (KA-1 domain) (Finnigan et al, 2016a,b). Thus, Hsl1 recruitment to the bud neck likely serves as a dual “sensor,” reporting that both septin collar assembly and the plasma membrane lipid composition have achieved the proper state to initiate the destruction of Swe1 and thereby license passage from G2 to M phase.…”

“…For certain proteins needed for AMR assembly, e.g., Bni5 (Lee et al, 2002), the septins continue to serve their scaffold function by mediating direct physical association with these factors, thereby anchoring and concentrating them at the bud neck (Patasi et al, 2015; Finnigan et al, 2015a, 2016a). For other proteins, e.g., Sec3 involved in localized deposition of secretory vesicles (Dobbelaere and Barral, 2004) and Chs2 involved in septum construction (Foltman et al, 2016), the two septin bands of the split collar seems to act like corrals and barriers to diffusion, thereby physically trapping these factors between them and thus confining them at this location indirectly (Dobbelaere and Barral, 2004; McMurray et al, 2011; Finnigan et al, 2016a). …”

“…In particular, at the bud neck, Dbf2-Mob1 phosphorylates another F-BAR protein, Hof1 (Meitinger et al, 2011), which also contains a C-terminal SH3 domain important for its function (Oh et al, 2013). Hof1 associates with septin structures throughout G1-S phase and while the AMR is assembled between the rings of the split septin collar primarily via direct interaction with Cdc10 (and Cdc12) (Vallen et al, 2000; Oh et al, 2013; Finnigan et al, 2016a). Association of Hof1 with the septins is mediated by a coiled-coil element in Hof1, and phosphorylation at a single Ser within this region by bud neck-localized Dbf2-Mob1 (an event that requires priming by prior phosphorylation of Hof1 by Cdk1 and Cdc5) promotes disassociation of Hof1 from the septin rings (Meitinger et al, 2013).…”

“…These rods can self-associate end-to-end to form filaments and can, depending in their subunit composition, also interact in other modes to form more elaborate super-structures, such as spirals, rings, braids, and gauze-like lattices (Rodal et al, 2005; Garcia et al, 2011; Oh and Bi, 2011; Bertin et al, 2012; Ong et al, 2014). Other factors nucleate the assembly of septin structures at discrete subcellular locations (Chen et al, 2011; Bi and Park, 2012) where they serve as both scaffolds (Shulewitz et al, 1999; Sakchaisri et al, 2004; Wloka et al, 2011; Bridges and Gladfelter, 2015) and diffusion barriers (Takizawa et al, 2000; Caudron and Barral, 2009) and thereby dictate, via direct and indirect interactions, the subcellular distribution of numerous other proteins (Gladfelter et al, 2001; McMurray and Thorner, 2009; Finnigan et al, 2016a). In particular, as discussed here, septin-based structures recruit, and thereby localize (and, in some cases, regulate the activity of) a multiplicity of protein kinases that integrate multiple inputs into signaling pathways and ultimately initiate ensuing biological responses (Figure 1).…”