Saccharomyces cerevisiae Gle2/Rae1 is involved in septin organization, essential for cell cycle progression

Abstract

Gle2/Rae1 is highly conserved from yeast to humans and has been described as an mRNA export factor. Additionally, it is implicated in the anaphase-promoting complex-mediated cell cycle regulation in higher eukaryotes. Here we identify an involvement for Saccharomyces cerevisiae Gle2 in septin organization, which is crucial for cell cycle progression and cell division. Gle2 genetically and physically interacts with components of the septin ring. Importantly, deletion of GLE2 leads to elongated buds, severe defects in septin-assembly and their cellular mislocalization. Septin-ring formation is triggered by the septin-regulating GTPase Cdc42, which establishes and maintains cell polarity. Additionally, activity of the master cell cycle regulator Cdc28 (Cdk1) is needed, which is, besides other functions, also required for G₂ /M-transition, and in yeast particularly responsible for initiating the apical-isotropic switch. We show genetic and physical interactions of Gle2 with both Cdc42 and Cdc28. Most importantly, we find that gle2∆ severely mislocalizes Cdc42, leading to defects in septin-complex formation and cell division. Thus, our findings suggest that Gle2 participates in the efficient organization of the septin assembly network, where it might act as a scaffold protein. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

Keywords: Gle2; Rae1; cell cycle regulation; mRNA export; septins.

Figure 1

Gle2 interacts with cell cycle regulators. (a) Synthetic genetic array (SGA) screen with essential temperature sensitive alleles reveals interactions of GLE2 with several groups functioning in cell cycle progression. A gle2∆ strain was crossed in an automated setup with each of the SGA strains and synthetic sickness or lethality was analysed. (b) Combination of gle2∆ with cell cycle mutants aggravates their growth defects, as visualized on agar plates in serial dilutions. (c) Gle2 interacts physically with several proteins involved in cell cycle regulation. Western blots showing co‐immunoprecipitations of myc‐Gle2 with GFP‐tagged versions of proteins involved in cell cycle progression. Rps3 served as a negative control.

Figure 2

Gle2 has a role in cell cycle regulation. (a) Deletion of GLE2 delays cell cycle progression. Flow cytometric analysis of wild type and gle2∆ cells after arrest with α‐factor (top). The percentage of cells with a haploid (1 N) or diploid (2 N) genome was calculated (bottom). (b) Deletion of GLE2 causes chromosome missegregation. Loss rates relative to wild type (top) and loss rates per cell division are depicted (bottom). mad1∆, defective in the spindle attachment checkpoint served as a positive control. (c) Nuclear mRNA export defects in gle2∆ are weak, when compared to the mRNA export mutant rat7–1 (nup159). Poly(A)⁺‐containing RNA was stained with a Cy3‐labelled oligo d(T)₅₀ probe (red); DNA was stained with Hoechst (blue) in fluorescence in situ hybridization experiments.

Figure 3

Gle2 is needed for correct formation of the septin ring. (a) Drop dilution test shows genetic interactions of gle2∆ with all septin mutants. (b) The temperature sensitive phenotype of the cdc10–1 mutant, regarding cell size and shape, is drastically enhanced when combined with a deletion of GLE2. (c) Quantification of the average cell length of the strains shown in (b). (d) Western blots of co‐immunoprecipitations (co‐IPs) show interactions of Cdc10 with Gle2. Rps3 served as a negative control. (e) Interaction of the septin ring components Cdc10 and Cdc11 is disturbed in gle2∆ cells as shown by western blots of co‐IPs between the septins. (f) Quantification of three different experiments shown in (e). (g) Cdc10‐GFP and Cdc11‐GFP are drastically mislocalized from the bud neck to the bud tip in strains deleted for GLE2. (h) Quantification of three different experiments shown in (g).

Figure 4

The cell cycle regulating GTPase Cdc42 requires Gle2 for correctly timed localization. (a) Drop dilution tests uncover genetic interaction of gle2∆ with mutant alleles of CDC42 and the major cell cycle kinase CDC28 (CDK1). (b) Co‐immunoprecipitation and western blot experiments reveal physical interaction of Cdc42 and Cdc28 with Gle2. (c) GFP‐microscopy during a time course experiment with synchronized cells show a prolonged presence of Cdc42 at the bud tip in gle2∆ cells. (d) Quantification of three different experiments shown in (c). A minimum of 100 cells was counted for each time point. (e) Average bud length of cells shown in (c) was determined and reveals significant elongation for cells lacking GLE2.