The Nim1 kinase Gin4 has distinct domains crucial for septin assembly, phospholipid binding and mitotic exit

Abstract

In fungi, the Nim1 protein kinases, such as Gin4, are important regulators of multiple cell cycle events, including the G2-M transition, septin assembly, polarized growth and cytokinesis. Compelling evidence has linked some key functions of Gin4 with the large C-terminal non-kinase region which, however, is poorly defined. By systematically dissecting and functionally characterizing the non-kinase region of Gin4 in the human fungal pathogen Candida albicans, we report the identification of three new domains with distinct functions: a lipid-binding domain (LBD), a septin-binding domain (SBD) and a nucleolus-associating domain (NAD). The LBD and SBD are indispensable for the function of Gin4, and they alone could sufficiently restore septin ring assembly in GIN4-null mutants. The NAD localizes to the periphery of the nucleolus and physically associates with Cdc14, the ultimate effector of the mitotic exit network. Gin4 mutants that lack the NAD are defective in spindle orientation and exit mitosis prematurely. Furthermore, we show that Gin4 is a substrate of Cdc14. These findings provide novel insights into the roles and mechanisms of Nim1 kinases in the regulation of some crucial cell cycle events.

Keywords: Candida albicans; Cdc14; Gin4; Nim1 kinases; Septin assembly.

Fig. 1.

CT1 and CT2 are indispensable for Gin4 function. (A) Schematic representation of CaGin4 domain organization and construction of truncation mutants. Numbers indicate amino acid residues. (B) Phenotype of gin4^CT1Δ-expressing cells (called JY8) under GIN4-ON and -OFF conditions. GIN4-ON cells were grown in MalMM and GIN4-OFF cells in GMM, respectively, at 30°C to log phase. Cells were examined by using differential interference contrast (DIC) and fluorescence microscopy. Arrows point to dots formed by GFP–Gin4^CT1Δ. (C) Phenotype of gin4^CT2Δ-expressing cells (called JY31). Cells were grown and examined as described in B. (D) Phenotype of gin4^CT3Δ-expressing cells (called JY49). For yeast cells, cells were grown as described in A. For hyphal growth, the yeast cells were induced for hyphal growth with 20% serum (FCS) at 37°C for 2 h. Scale bars: 5 µm.

Fig. 2.

CT1 contains the domains required for phospholipid binding and localization to the plasma membrane. (A) Phenotype of CT1-expressing cells (called JY9). Yeast and hyphal cells were grown and examined as described in Fig. 1. Arrows point to septin rings at the junction between a new bud and the mother cell. (B) CT1.3 (residues 1151–1250) mediates the localization at the plasma membrane. CT1 was truncated into smaller fragments, each tagged with GFP at the N-terminus and expressed in strain LCR43. All seven basic residues were replaced with serine in CT1.3 to yield CT1.3_7S (called JY23). (C) Purified GST–CT1 and GST–CT1.3 bind to phosphoinositides in vitro. GST, GST–CT1 and GST–CT1.3 expressed and purified from E. coli were tested for their ability to bind phospholipids by using the PIPstrips™. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanomaline; PS, phosphatidylserine; S1P, sphingosine 1-phosphate. Scale bars: 5 µm.

Fig. 3.

CT2 localizes to the bud neck and interacts with septins. (A) CT2 and two sub-fragments (2.1 and 2.2) tagged with N-terminal GFP were expressed in gin4Δ/P_MAL2-GIN4 cells that coexpressed Cdc12–mCherry (called JY35, JY37 and JY39, respectively). Cells were grown in GIN4-ON conditions at 30°C for microscopy analysis. (B) Coimmunoprecipitation of CT2 with Cdc12. C-terminally Myc-tagged Cdc12 was expressed in gin4Δ/P_MAL2-GIN4 cells that coexpressed GFP–CT2 (called JY40); gin4Δ/P_MAL2-GIN4 cells that expressed Cdc12–Myc (called JY69) alone were included as a negative control. Middle panel, cells were grown under GIN4-ON or -OFF conditions at 30°C to log phase. Coimmunoprecipitation was performed with anti-Myc beads and washed with buffer containing 150 mM NaCl, followed by western blot analysis to detect GFP or Myc. Right panel, immunoprecipitated anti-Myc beads were washed with buffer containing 1 M NaCl before western blot analysis. (C) Purified GST–CT2 pulls down Cdc12 from C. albicans cell lysates. GST–CT2 that had been purified from E. coli was incubated with GST-Trap beads. GST-Trap beads with or without bound GST–CT2 were mixed and incubated with C. albicans cell lysates expressing Cdc12–Myc (JY69). After washes with buffer containing 150 mM or 1 M NaCl, proteins on the beads were probed for Myc or GST by western blotting. Scale bars: 5 µm.

Fig. 4.

The CT2+1 fragment can restore septin ring formation and localization to the bud neck in GIN4-OFF yeast cells. (A) Cells expressing Cdc12–mCherry (called JY70) were cultured overnight under GIN4-ON conditions at 30°C. Elutriated G1 cells were released into GIN4-ON or GIN4-OFF conditions. Cells at different stages of budding were harvested for microscopy analysis. (B) G1 cells that coexpressed Cdc12–mCherry and GFP–CT2+1 (called JY71) were prepared as described above and released into GIN4-OFF conditions for further growth. Cells at different stages of bud growth (upper panel) and of an overnight culture (lower panel) were harvested for microscopy. (C) Cells that coexpressed Myo1–mCherry and GFP–CT2+1 (called JY73) were grown under GIN4-ON or GIN4-OFF conditions at 30°C to log phase before microscopy analysis. Arrows point to Myo1 at the bud neck. (D) Cells that coexpressed Cdc12–mCherry with GFP–Gin4 (called JY30) or GFP–CT2+1 (called JY71) were grown overnight under GIN4-OFF conditions before shifting to GMM+20% FCS for hyphal induction at 37°C for 2 h. (E) The same cells that are described in D after 3.5 h of hyphal induction. (F) FRAP analysis of the septin ring in cells that expressed WT Gin4 (JY70, n=13) or CT2+1 as the sole Gin4 source (JY71, n=12). Cells with a medium-sized bud were selected for FRAP, and the entire septin ring was bleached. Fluorescence recovery was measured at 5-min intervals after photobleaching. Average values were used to generate the curves. Scale bars: 5 µm.

Fig. 5.

CT3 contains a domain for localization to the nucleolus. (A) GIN4-OFF gin4^CT3Δ cells exhibited a multi-nucleated phenotype. Yeast cells of strain JY49 that had been grown under GIN4-OFF conditions were DAPI-stained to visualize the nuclei. (B) Time-lapse microscopy examination of the spindle in GIN4-OFF gin4^CT3Δ cells. Tem1 was C-terminally tagged with mCherry in gin4Δ/P_MAL2-GIN4 (called JY51) and gin4Δ/P_MAL2-GIN4 gin4^CT3Δ (called JY50) cells. A representative GIN4-ON (upper panel) cell and a GIN4-OFF gin4^CT3Δ cell (JY50) are shown (time, h:min:sec). (C) CT3 localizes to the nucleus. GIN4-ON cells expressing GFP–CT3 (called JY43) were stained with DAPI before microscopy analysis. (D) Time-lapse microscopy examination of GFP–CT3 localization during a cell cycle in GIN4-ON GFP–CT3 cells (JY43). (E) Truncation of CT3 in order to locate the domain responsible for nucleolus foci localization. CT3 was truncated into CT3.1 (residues 451–650), CT3.2 (residues 451–550) and CT3.3 (residues 551–650). Each fragment was tagged with GFP at the N-terminus and expressed in gin4Δ/P_MAL2-GIN4 cells (called JY53, JY54 and JY55, respectively). Cells were grown in GIN4-ON conditions and examined with fluorescence microscopy. (F) CT3.3 colocalizes with Nop1. Nop1 was tagged with mCherry in cells that coexpressed GFP–CT3, GFP–CT3.3 and GFP–Gin4^CTΔ (called JY58, JY59 and JY60, respectively). Scale bars: 5 µm.

Fig. 6.

Cdc14 physically associates with the NAD and dephosphorylates Gin4. (A) Colocalization of CT3 and Cdc14. Cdc14–GFP was expressed from its endogenous promoter in gin4Δ/P_MAL2-GIN4 cells that coexpressed mCherry–CT3 (called JY62) or mCherry–Gin4^CT1Δ (called JY63). Cells were grown in GIN4-ON conditions and examined by performing fluorescence microscopy. (B) The NAD coimmunoprecipitates with Cdc14. Cdc14 was Myc-tagged C-terminally in gin4Δ/P_MAL2-GIN4 cells that coexpressed GFP–Gin4, GFP–CT3, GFP–CT3.3 and GFP–Gin4^CTΔ (called JY64, JY65, JY66 and JY67, respectively). Coimmunoprecipitation was performed in GIN4-ON cells by pulling down proteins using anti-GFP beads, followed by western blot analysis of the precipitates for Myc or GFP. (C) Cdc14 can dephosphorylate Gin4 in vitro. GST–Cdc14 and GST–Cdc14^C275S were purified from E. coli and mixed with immunopurified GFP–Gin4 (JY56) for in vitro phosphatase assays. Gin4 and phosphorylated Gin4 in the reaction products were detected by western blotting for GFP and PScdk, respectively. A λ-phosphatase-treated (λpp) sample was included as a positive control. (D) Involvement of Cdc14 in Gin4 dephosphorylation in vivo. Gin4–GFP was expressed from its endogenous promoter in WT (BWP17) and cdc14Δ/Δ cells that coexpressed Nop1–mCherry (called JY72 and JY68, respectively). Stationary-phase cultures were released into fresh medium for synchronous growth. Aliquots were collected at timed intervals for microscopy evaluation of cell cycle progression (n>100, upper panel). The rest of the cells were subjected to immunoprecipitation of GFP and western blot analysis for PScdk or GFP to reveal phosphorylated Gin4 and total Gin4–GFP (lower panel). Phosphorylated-Gin4–GFP signal (upper band in bottom blot) was quantified through analysis with ImageJ and normalization against the Gin4–GFP signal. The level of phosphorylated-Gin4–GFP (Phos-Gin4) at each time point is shown under the blot. Scale bars: 5 µm.

Fig. 7.

A schematic description of the function of Gin4 during a cell cycle.