Ash1, a daughter cell-specific protein, is required for pseudohyphal growth of Saccharomyces cerevisiae

Abstract

Ash1 (for asymmetric synthesis of HO) was first uncovered in genetic screens that revealed its role in mating-type switching. Ash1 prevents HO expression in daughter cells. Because Ash1 has a zinc finger-like domain related to that of the GATA family of transcription factors, it presumably acts by repressing HO transcription. Nonswitching diploid cells also express Ash1, suggesting it could have functions in addition to regulation of HO expression. We show here that Ash1 has an essential function for pseudohyphal growth. Our epistasis analyses are consistent with the deduction that Ash1 acts separately from the mitogen-activated protein kinase cascade and Ste12. Similarly to the case in yeast form cells, Ash1 is asymmetrically localized to the nuclei of daughter cells during pseudohyphal growth. This asymmetric localization reveals that there is a previously unsuspected daughter cell-specific function necessary for pseudohyphal growth.

FIG. 1

Role of Ash1 in filamentous growth. (A) Invasive growth. Haploid ASH1 [SC114(pRS426)], ash1Δ [SC112(pRS426)], and 2μm ASH1 [SC112(pAS163)] strains were grown on a YPD plate for 5 days. Photographs show patches before and after the plate was washed with water. (B) Pseudohyphal-colony formation. Diploid ASH1/ASH1 [L5783(pRS426)], ash1Δ/ash1Δ [SC125(pRS426)], and 2μm ASH1 [SC125(pAS163)] strains were streaked out on nitrogen starvation medium (SLAD) and grown for 2.5 days at 30°C. Photographs show representative colonies of each strain.

FIG. 2

Effects of Ash1 overexpression on pseudohyphal-colony formation in strains with deletions of MAPK activation pathway components. (A) ste20Δ/ste20Δ (HLY492), ste11Δ/ste11Δ (HLY506), ste7Δ/ste7Δ (HLY351), and ste12Δ/ste12Δ (HLY352) diploid strains containing either vector (pRS426), 2μm ASH1 (pAS163), or 2μm STE12 (pNC248) were grown on SLAD. (B) The ste20Δ/ste20Δ strain (HLY492) containing either vector (pRS426) or GAL-ASH1 (pNC543) was streaked out on nitrogen starvation medium containing galactose. Photographs show representative colonies of each strain after 2 (A) or 3 (B) days of growth at 30°C.

FIG. 3

Comparison of ASH1 and ash1Δ mutant strains for pseudohyphal-colony formation promoted by hyperactivation of the pathway. (A) ASH1/ASH1 (L5783) and ash1Δ/ash1Δ (SC125) diploid strains containing 2μm STE12 (pNC248) were grown on SLAD for 2 days at 30°C. (B) ASH1/ASH1 (L5783) and ash1Δ/ash1Δ (SC125) diploid strains containing GAL-STE11^296–717 (pNC544) were grown on nitrogen starvation medium containing galactose for 1 day at 30°C. Photographs are of representative colonies.

FIG. 4

Synthetic pseudohyphal-colony phenotype of ash1Δ and ste12Δ mutants. ASH1/ASH1 STE12/STE12 [L5783(pRS426)], ash1Δ/ash1Δ STE12/STE12 [SC125(pRS426)], ASH1/ASH1 ste12Δ/ste12Δ [HLY352(pRS426)], and ash1Δ/ash1Δ ste12Δ/ste12Δ [SC137(pRS426)] diploid strains were patched onto SLAD and grown at 30°C for 4 days. Photographs show a representative region of an edge from each patch.

FIG. 5

Comparison of ash1Δ and ste12Δ single- or double-mutant strains for pseudohyphal-colony formation promoted by hyperactivation of the pathway. ASH1/ASH1 STE12/STE12 (L5783), ash1Δ/ash1Δ STE12/STE12 (SC125), ASH1/ASH1 ste12Δ/ste12Δ (HLY352), and ash1Δ/ash1Δ ste12Δ/ste12Δ (SC137) diploid strains containing either RAS2-V¹⁹ (pMW2) (A) or 2μm PHD1 (pCG37) (B) were grown on SLAD for 2 days at 30°C. Photographs are of representative colonies.

FIG. 6

Localization of GFP-Ash1 during pseudohyphal growth. Fluorescent (A and C) and differential interference contrast (B and D) views of cells are shown. (A and B) Diploid strain SC126, which is heterozygous for GFP-ASH1, grown on SLAD for 12 h. Insets show a mother-daughter pair of yeast form cells of the same diploid strain grown on SD-Ura. (C and D) Diploid ash1Δ/ash1Δ strain SC125 with pNC513, which expresses GFP-ASH1 from the 2μm vector pRS426, grown on SLAD for 12 h. m, mother cell; d, daughter cell.

FIG. 7

Model for the pseudohyphal-response pathway. The diagram shows the postulated relationship of signal transmission components that function after the starvation signal(s) for induction of pseudohyphal growth. Arrows indicate activation or stimulation. Lines with bars indicate repression or inhibition. See the text for discussion of the evidence suggesting these relationships.