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

Septin proteins contribute to many eukaryotic processes involving cellular membranes. In the budding yeast Saccharomyces cerevisiae, septin hetero-oligomers interact with the plasma membrane (PM) almost exclusively at the future site of cytokinesis. While multiple mechanisms of membrane recruitment have been identified, including direct interactions with specific phospholipids and curvature-sensitive interactions via amphipathic helices, these do not fully explain why yeast septins do not localize all over the inner leaflet of the PM. While engineering an inducible split-yellow fluorescent protein (YFP) system to measure the kinetics of yeast septin complex assembly, we found that ectopic co-overexpression of two tagged septins, Cdc3 and Cdc10, resulted in nearly uniform PM localization, as well as perturbation of endogenous septin function. Septin localization and function in gametogenesis were also perturbed. PM localization required the C-terminal YFP fragment fused to the C terminus of Cdc3, the septin-associated kinases Cla4 and Gin4, and phosphotidylinositol-4,5-bis-phosphate (PI[4,5]P2 ), but not the putative PI(4,5)P2 -binding residues in Cdc3. Endogenous Cdc10 was recruited to the PM, likely contributing to the functional interference. PM-localized septins did not exchange with the cytosolic pool, indicative of stable polymers. These findings provide new clues as to what normally restricts septin localization to specific membranes.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins.

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 nonnative conformations and formed nonnative homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble posttranslationally into native complexes. One septin, Cdc12, was so dependent on cotranslational 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, cotranslational assembly pathway in which general cytosolic chaperones act as key intermediaries.