Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins

Abstract

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted non-native conformations and formed non-native homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble post-translationally into native complexes. One septin, Cdc12, was so dependent on co-translational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, co-translational assembly pathway in which general cytosolic chaperones act as key intermediaries.

Simultaneous co‐overexpression of Saccharomyces cerevisiae septins Cdc3 and Cdc10 drives pervasive, phospholipid‐, and tag‐dependent plasma membrane localization

“…We quantified total cellular fluorescence in wild‐type haploid cells at time points following induction and observed a gradual increase in signal, indicative of transcription, translation, folding, and assembly of the tagged Cdc3 and Cdc10, and reconstitution and maturation of the split fluorophore (Figure 1a,b). The kinetics of accumulation (<2‐fold over 7 h) were considerably slower than for GFP‐tagged Cdc3, for which fluorescence increased >4‐fold in 6 h, even though here we used a higher concentration of galactose (Hassell et al, 2022; Schaefer et al, 2016).…”

“…Individual overexpression of tagged wild‐type Cdc3 or Cdc10 results in septin ring signal plus accumulation diffusely throughout the cytoplasm/nucleus (Hassell et al, 2022; Johnson et al, 2015). Unexpectedly, and especially after long induction times, BiFC signal was often found extensively along the PM (Figure 2a).…”

“…The excess septin––super‐stoichiometric to the endogenous septins––remained diffusely localized in the cytoplasm/nucleus, ostensibly “waiting” until newly‐synthesized partner septins were synthesized and, via de novo folding, became available for new complex assembly (Hassell et al, 2022). In the meantime, the excess septins were kept assembly‐competent by specific cytosolic chaperones, which we proposed to “protect” the excess septins' dimerization interfaces from inappropriate interactions (Hassell et al, 2022). In particular, our data pointed to chaperone occupancy of the “G” interface that surrounds and encompasses each septin's guanine nucleotide‐binding pocket (Hassell et al, 2022; Johnson et al, 2015; Sirajuddin et al, 2007).…”

“…In the meantime, the excess septins were kept assembly‐competent by specific cytosolic chaperones, which we proposed to “protect” the excess septins' dimerization interfaces from inappropriate interactions (Hassell et al, 2022). In particular, our data pointed to chaperone occupancy of the “G” interface that surrounds and encompasses each septin's guanine nucleotide‐binding pocket (Hassell et al, 2022; Johnson et al, 2015; Sirajuddin et al, 2007). If, rather than having to wait for them, plenty of molecules of the G‐dimer partner are immediately available, then two partner septins simultaneously in excess to the others––and in 1:1 stoichiometry to each other––could rapidly outcompete the chaperones to form a stable dimer.…”

“…Quantification of septin ring fluorescence is used as a proxy for successful incorporation of the GFP-tagged septin into polymerization-competent hetero-oligomers, whereas signal in the cytoplasm and nucleus is interpreted as representing septin-GFP molecules that have not yet incorporated into hetero-oligomers plus those that did achieve native assembly but did not polymerize into filaments. By measuring the kinetics of accumulation of septin ring signal relative to cytoplasm/nucleus signal at time points following the addition of galactose, we successfully applied this technique to identify both septin mutations (Schaefer et al, 2016) and chaperone mutations (Hassell et al, 2022) that slow septin folding/assembly. Here we sought to streamline this assay by eliminating the need to determine the subcellular localization of fluorescence signal.…”