@article {Johnson744722, author = {Courtney R. Johnson and Marc G. Steingesser and Andrew D. Weems and Anum Khan and Aur{\'e}lie Bertin and Amy Gladfelter and Michael A. McMurray}, title = {Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast}, elocation-id = {744722}, year = {2019}, doi = {10.1101/744722}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Septin proteins co-assemble into hetero-oligomers that polymerize into cytoskeletal filaments with a variety of cellular functions. In Saccharomyces cerevisiae, where septins were first discovered, five subunits comprise two species of septin hetero-octamers, Cdc11/Shs1{\textendash}Cdc12{\textendash}Cdc3{\textendash}Cdc10{\textendash}Cdc10{\textendash}Cdc3{\textendash}Cdc12{\textendash}Cdc11/Shs1. Septins evolved from ancestral GTPases. We previously found evidence that slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1 into septin complexes. It was unclear why many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that the small molecule guanidine hydrochloride (GdnHCl) rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1{\textendash}Cdc12{\textendash}Cdc3{\textendash}Cdc3{\textendash}Cdc12{\textendash}Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium ion (Gdm) occupies the site of a {\textquotedblleft}missing{\textquotedblright} Arg sidechain that is present in other fungal species in which (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers co-exist with octamers in wild-type cells. These findings support a model in which Gdm reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to hetero-octamers. Given recent reports that septin hexamers made natively in human cells also involve bypass of Cdc10-like central subunits via homodimerization of an active GTPase, our results provide new mechanistic details that likely apply to septin assembly throughout phylogeny.AUTHOR SUMMARY Life requires proper assembly of multi-subunit protein oligomers. The subunits of many oligomers evolved by gene duplication from a single ancestral protein. Specialization of inter-subunit interfaces ensures that subunit organization within these hetero-oligomers is non-random, but exactly how is often unclear. Hetero-oligomers of septin proteins perform a variety of cellular functions in nearly all eukaryotes. Subunit positioning within septin oligomers can often be predicted from sequence relatedness, yet the number and identity of subunits varies by organism and even within a single cell, raising the question of how the rules governing septin oligomer assembly can be simultaneously flexible and strict. We previously found that a septin{\textquoteright}s ability to hydrolyze the nucleotide GTP allows flexibility. Here we serendipitously discovered that a ten-atom molecule, guanidinium, appears to bind in vivo to a GTPase-dead septin where it promotes flexibility in oligomerization by mimicking the presence of an arginine sidechain found in active GTPases. Our findings suggest that loss of GTPase activity during evolution constrained septin oligomerization pathways. In addition to elucidating a mechanism that may be relevant to other oligomeric GTPases, the ability of guanidinium to rescue function in vivo of proteins lacking specific arginines has both evolutionary and therapeutic implications.}, URL = {https://www.biorxiv.org/content/early/2019/08/24/744722}, eprint = {https://www.biorxiv.org/content/early/2019/08/24/744722.full.pdf}, journal = {bioRxiv} }