Regulation of phosphoinositide levels by the phospholipid transfer protein Sec14p controls Cdc42p/p21-activated kinase-mediated cell cycle progression at cytokinesis

Abstract

Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade.

FIG. 1.

Suppression of growth of sec14^ts cki1 cells. (A) sec14^ts cki1 cells expressing the indicated genes from a high-copy-number plasmid were grown to log phase in culture at 25°C. Equal numbers of cells were plated in 1:10 serial dilutions onto minimal medium and incubated at 25°C or 37°C. (B) CLA4 and CDC42 also suppress cki1 bypass of sec14^ts. (C) sec14^ts cki1 cells expressing the indicated genes from a high-copy-number plasmid were transformed with a low-copy-number plasmid containing SEC14 and grown to log phase in culture at 25°C. Equal numbers of cells were plated in 1:10 serial dilutions onto minimal medium and incubated at 25°C or 37°C.

FIG. 2.

PAKs do not affect the growth of sec14^ts cells. (A) The sec14^ts strain was transformed with a high-copy-number plasmid expressing CLA4 or STE20 or with an empty vector, and equal numbers of cells were serially diluted and grown on solid medium at the indicated temperatures. (B) The sec14^ts cki1 strain was transformed with a low-copy-number plasmid containing CKI1 or an empty vector, followed by a second transformation with a low-copy-number plasmid containing an empty vector or SEC14 and a high-copy-number plasmid containing CLA4 or an empty vector. Cells were grown to mid-log phase at 25°C, and equal numbers of cells were serially diluted, spotted onto solid medium, and grown at 25°C or 37°C.

FIG. 3.

PAK enzyme activity is required for growth suppression. (A) The sec14^ts cki1 strain expressing STE20 or a version of STE20 (K649R) that renders the kinase inactive from a high-copy-number plasmid was grown to log phase in liquid culture at 25°C. Equal numbers of cells were plated in 1:10 serial dilutions onto minimal medium and incubated at 25°C or 37°C. (B and C) The sec14^ts cki1 strain was transformed with 2μm plasmids for overexpression of ARL1, ARF1, ARF2, or BOI1. Cells were grown to log phase in liquid culture at 25°C. Equal numbers of cells were plated in 1:10 serial dilutions onto minimal medium and incubated at 25°C, 35°C, or 37°C.

FIG. 4.

Golgi apparatus-derived vesicular transport and viability of sec14^ts cki1 cells. (A) Invertase secretion indices of wild-type, sec14^ts, and sec14^ts cki1 cells with or without high-copy-number CLA4, STE20, or CDC42 were determined. Error bars, standard errors of the means (n = 3). (B) Yeast cells were spotted in 1:10 dilutions of equal cell numbers onto a medium with or without 20 μg/ml calcofluor white. (C) Strains were grown to early log phase at 25°C, diluted to equal cell numbers, and shifted to 37°C. At the indicated time points, equal cell numbers were removed, and 1:10 serial dilutions were spotted onto plates and incubated at 25°C.

FIG. 5.

Suppressed strains accumulate large buds and multiple buds but have normal septin localization. (A) CTY160 (sec14^ts cki1) cells containing a vector control or high-copy-number CLA4, STE20, or CDC42 were quantified according to bud size, and cells with different bud sizes are represented as percentages of the total population. (B) Calcofluor white staining of chitin at the bud neck. CTY160 cultures with a vector or overexpressing CLA4, STE20, or CDC42 were grown to log phase at 25°C, shifted to 37°C for 15 h, and then stained with calcofluor white. (C) CTY160 cells with or without high-copy-number CLA4, STE20, or CDC42 were grown to log phase at 25°C and then shifted to 37°C for 15 h. Cultures were fixed, and DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI) to visualize the nucleus. (D) CTY160 cells containing an empty vector or high-copy-number CLA4, STE20, or CDC42 were grown to log phase at 25°C and then shifted to 37°C for as long as 15 h. The septin Cdc12-GFP was localized to the bud neck in both suppressed and vector control strains at 37°C. Typical images are shown.

FIG. 6.

Cdc42p/PAK does not suppress the growth of cells lacking KES1. (A) Strains CTY100 (sec14^ts sac1) and CTY159 (sec14^ts kes1) were transformed with multicopy CLA4, STE20, or CDC42. Strains were grown to log phase, and equal cell numbers were spotted in 1:10 serial dilutions onto minimal medium plates at 25°C and 37°C. (B) sec14^ts cki1 or sec14^ts kes1 cells were transformed with a low-copy-number plasmid encoding GFP-CDC42. Cells were grown to mid-log phase at 25°C, and a subset of these cells were shifted to 37°C for 1 h prior to imaging of live cells. Typical images are shown.

FIG. 7.

Phosphoinositides relieve growth suppression by Cdc42p/PAKs. (A) sec14^ts cki1 cells (CTY160) transformed with 2μm plasmids expressing CLA4, STE20, or CDC42 were transformed with a high-copy-number plasmid containing PIK1, STT4, or MSS4 or with an empty vector and then grown to log phase in liquid culture at 25°C. Equal cell numbers were plated in 1:10 serial dilutions onto minimal medium plates and incubated at 25°C or 37°C. (B) The cdc42^ts strain was transformed with high-copy-number plasmids expressing PIK1, STT4, MSS4, SEC14, or an empty vector and was grown to log phase in liquid culture at 25°C. Equal cell numbers were plated in 1:10 serial dilutions onto minimal medium plates and incubated at 25°C, 35°C, or 37°C.