Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

Abstract

Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3–Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium occupies the site of a ‘missing’ Arg side chain found in other fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers natively co-exist with octamers. We propose that guanidinium reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to octamers. Since homodimerization by a GTPase-active human septin also creates hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply throughout phylogeny.

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

“…1 B ). We predicted that arg3(R69G) mutant yeast would be auxotrophic for Arg and that this auxotrophy would be rescued by the addition of 3 mM GdnHCl to the medium, a concentration that rescues cdc10 mutants (19). We engineered the R69G substitution in a plasmid-encoded copy of ARG3 and introduced the mutant plasmid or its WT parent into arg3 Δ cells.…”

“…We were inspired to investigate the potential of GdnHCl for chemical rescue in living cells by our prior, serendipitous discovery that GdnHCl reverses the TS phenotypes of S. cerevisiae cells carrying mutations in the CDC10 gene (19). CDC10 encodes a subunit of the essential yeast septin protein complex.…”

“…Our data indicate that, rather than binding to mutant Cdc10 molecules, Gdm binds to WT molecules of another septin protein in the same complex, at the former site of an Arg side chain that was “lost” during fungal evolution. In many yeast species, loss of the Arg and of GTPase activity by the same septin constrained the kinds of septin hetero-oligomers that are able to assemble in those cells (19). Specifically, we think that Gdm alters the conformation of the homodimerization interface that encompasses the GTP-binding pocket in a way that promotes bypass of Cdc10 subunits during septin complex assembly (19).…”

“…In many yeast species, loss of the Arg and of GTPase activity by the same septin constrained the kinds of septin hetero-oligomers that are able to assemble in those cells (19). Specifically, we think that Gdm alters the conformation of the homodimerization interface that encompasses the GTP-binding pocket in a way that promotes bypass of Cdc10 subunits during septin complex assembly (19). Restoring the “missing” Arg residue was insufficient to recapitulate the effect of GdnHCl, but it blocked the ability of GdnHCl to rescue, consistent with a model in which multiple amino acid changes during evolution disfavored Cdc10 bypass, and Gdm acts as a “chemical chaperone” to influence folding of a single septin protein in ways that cannot be predicted by protein sequence alone.…”

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1

“…Nonetheless, this reported complementation presumably requires that SEPT6 (and SEPT10) be able to form a functional NC homodimer that is able to engage at its flanks yeast Cdc3 via a G interface, highlighting the incredible flexibility inherent in septin–septin interaction. In this same regard, it has been inferred that the (obligate) inclusion of Cdc10 at the central position within the yeast hetero-octamer may have been coupled to the loss of the ability of the Cdc3 subunit to hydrolyze its bound GTP, an event that seems to have occurred prior to the split between the yeast genera Saccharomyces , Ashbya , and Kluyveromyce ( Johnson et al 2020 ). Indeed, biochemical studies of the corresponding human proteins ( Zent and Wittinghofer 2014 ) demonstrate that SEPT9, like yeast Cdc10, is GTPase competent, whereas the flanking septin, SEPT7, like yeast Cdc3, lacks the capacity to hydrolyze its bound GTP.…”