Lean forward: Genetic analysis of temperature‐sensitive mutants unfolds the secrets of oligomeric protein complex assembly

Abstract

Multisubunit protein complexes are essential for cellular function. Genetic analysis of essential processes requires special tools, among which temperature-sensitive (Ts) mutants have historically been crucial. Many researchers assume that the effect of temperature on such mutants is to drive their proteolytic destruction. In fact, degradation-mediated elimination of mutant proteins likely explains only a fraction of the phenotypes associated with Ts mutants. Here I discuss insights gained from analysis of Ts mutants in oligomeric proteins, with particular focus on the study of septins, GTP-binding subunits of cytoskeletal filaments whose structures and functions are the subject of current investigation in my and many other labs. I argue that the kinds of unbiased forward genetic approaches that generate Ts mutants provide information that is largely inaccessible to modern reverse genetic methodologies, and will continue to drive our understanding of higher-order assembly by septins and other oligomeric proteins.

An extended loop in CE7 carbohydrate esterase family is dispensable for oligomerization but required for activity and thermostability

“…It is well recognized that quaternary structure of proteins have evolved to optimize intrinsic flexibility and dynamics at multiple spatial and timescales, without disruption of stability and functional activity (Devenish and Gerrard, 2009;Marsh, 2013;Marsh and Teichmann, 2014;Nishi et al, 2013). Indeed, higher order oligomerization is among the many adaptations that allow proteins to maintain structural stability under different conditions (Kumar et al, 2000;McMurray, 2014;Reed et al, 2013;Russell and Taylor, 1995;Tanaka et al, 2004).…”

Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast

“…, 2008 ; Weems et al. , 2014 ) at positions that are predicted to be relatively rigid/immobile within the folded structure ( McMurray, 2014 ) and probably bias the conformational equilibria such that native residues (especially those normally buried) adopt non-native positions.…”

Septin Mutations in Human Cancers

“…Septins, however, contain additional helices (α0, α5′, and α6) and β-strands (e.g., β7, β8), which in part support tandem dimerization into oligomers and polymers (Sirajuddin et al, 2007, 2009; Macedo et al, 2013). Assembly of these septin heteromers depends on GTP-binding and hydrolysis, which further stabilizes the dimerization interfaces through allosteric effects (McMurray, 2014; Zent and Wittinghofer, 2014; Zeraik et al, 2014). In contrast to the monomeric Ras GTPases, whose function relies on the hydrolysis and exchange of GTP by GTPase activating proteins (GAPs) and guanine exchange factors (GEFs), septins hydrolyze and exchange GTP on their own, albeit at very slow rates (Sheffield et al, 2003; Vrabioiu et al, 2004; Huang et al, 2006; Abbey et al, 2016).…”