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

Abstract

Septin proteins hetero-oligomerize to form filaments. Mutant septins that subtly misfold a key oligomerization interface retain some function when expressed alone but are excluded from filaments when the wild-type allele is present. Cytosolic chaperones mediate this “quality control” via prolonged interactions with the mutant polypeptides.

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

“…We previously used incorporation into the septin rings at the bud neck as an indirect readout of oligomerization competence, quantified by line scans across the bud necks of cells expressing a fluorescently-tagged septin. 15 To introduce a kinetic component to this experiment, we reasoned that the kinetics of bud neck accumulation following induction of expression should be directly related to the kinetics of septin translation, folding, and hetero-oligomerization. Cdc3-GFP was expressed at room temperature (»22 C) under control of the galactose-inducible GAL1/10 promoter from a low-copy plasmid in cells co-expressing untagged, WT Cdc3 from the CDC3 promoter at its endogenous locus.…”

“…14 We further found that, when a wild-type (WT) allele of the same septin is available and the supply of other septin subunits is limiting, these same TS mutant septin proteins are subject to chaperone-dependent exclusion from higher-order assemblies, even at temperatures permissive for function. 15 We concluded that the WT septin folds faster into an oligomerization-competent form, whereas the mutant septin is slow to adopt conformations that are no longer recognized as misfolded by cytosolic chaperones, and is thus delayed in becoming available for septin-septin oligomerization. 15 Inherent in this "quality control" (QC) model is the idea that there must be a transient "window" of time and/or space that is permissive for septin heterooligomerization, and WT septins are more successful than G interface mutants at populating this interval.…”

“…15 We concluded that the WT septin folds faster into an oligomerization-competent form, whereas the mutant septin is slow to adopt conformations that are no longer recognized as misfolded by cytosolic chaperones, and is thus delayed in becoming available for septin-septin oligomerization. 15 Inherent in this "quality control" (QC) model is the idea that there must be a transient "window" of time and/or space that is permissive for septin heterooligomerization, and WT septins are more successful than G interface mutants at populating this interval. Outside the "window of opportunity," a nascent septin polypeptide hetero-oligomerizes with much reduced efficiency, and polypeptides that have "missed the window" cannot simply wait for it to "re-open" -this is a once-in-a-lifetime opportunity.…”

“…The basic requirements for KADO, illustrated in Figure 5, are: (i) a stoichiometric complex whose subunits are present in equivalent amounts (i.e., oligomerization partners are always "limiting"); (ii) an oligomerization pathway that proceeds via steps that are rapid and effectively irreversible (i.e., subcomplexes are transient assembly intermediates, and may be, as is the case for septins, 15,[36][37][38] undetectable in WT cells); (iii) a finite "window of opportunity" -spatial and/or temporal-for de novo oligomerization whose closure prevents unassembled subunits from participating in subsequent assembly events (destruction of unincorporated subunits represents a common and efficient way to "inactivate" such molecules); and (iv) different alleles of a subunit with distinct abilities to adopt an oligomerization-competent conformation. In principle, KADO does not require a role for chaperones in influencing the kinetics of maturation of nascent polypeptides, but we suspect that most slow-maturing polypeptides will become targets for prolonged engagement by chaperones if, for example, they struggle to bury hydrophobic residues.…”

“…If only a single allele is expressed, or if the other septin subunits are in excess, then a slow-folding mutant is capable of occupying available subunit positions, at least when the temperature is kept low. 15 Increasing the temperature upregulates chaperones and presumably exacerbates folding defects, causing more mutant molecules to "miss the window," and causing cells expressing only the TS mutant allele to die.…”