Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules

Abstract

Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here we used a combination of targeted and unbiased approaches to look for other cases of “chemical rescue” by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from “off-target” effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued ∼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.

Biocontainment Techniques and Applications for Yeast Biotechnology

“…Similar works on bacterial synthetic auxotrophs have been made, including phosphite-dependent Synechococcus elongatus [98] and Pseudomonas putida [99]. In S. cerevisiae it has been identified a series of mutations in CDC10 gene, which codify an essential septin protein that can be rescued in the presence of small molecules, like guanidinium ion [100,101]. Although the authors of this work have not applied the CDC10 conditional mutants for biocontainment, the data may indicate new techniques based on chemical rescue for GMY biocontainment.…”

Evolutionary degeneration of septins into pseudoGTPases: impacts on a hetero-oligomeric assembly interface

“…For example, a huge increase in the concentration of intracellular trehalose during yeast sporulation ( Roth, 1970 ) likely stabilizes proteins and protects against aggregation ( Singer and Lindquist, 1998 ; Jain and Roy, 2009 ). We previously demonstrated how other naturally occurring small molecules like guanidine and trimethylamine N-oxide can restore viability to septin-mutant yeast cells with single substitutions in canonical pocket residues ( Johnson et al, 2020 ; Hassell et al, 2021 ). Temperature also has a tremendous impact on the functional consequences of changes to septin G interface contacts.…”