Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth

Abstract

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different beta-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.

FIG. 1.

Assay for invasive growth. Cells were grown on solid CSM plus 0.1% glucose (A and B) or on YEP plus 2% glucose containing 2.5% agar (B) for 2 days at 26°C and photographed. Plates were washed and photographed again. The wild-type strain was MCY4565 and mutant strains (listed in Table 1) were also all MATa and carried the same auxotrophic markers (in particular, all are Ura⁺), so that invasiveness would not be affected by differences in genetic background. On YEP plus 2% glucose, invasive growth of the wild type is easily detectable by 3 days.

FIG. 2.

Assay for adherence to plastic. Cells were assayed for adherence to polystyrene (36) as described in Materials and Methods. Cells were resuspended in CSM with 2 or 0.1% glucose, as indicated, and transferred to the wells of a microtiter plate. (A) Strains were MCY4702, MCY4704, and MCY4712; all were MATa. Cells were incubated for the indicated time before washing. (B) The wild-type strain was MCY4565, and mutant strains were also all MATa and carried the same auxotrophic markers. Cells were incubated for 7.5 h before washing. Assays were carried out in quadruplicate.

FIG. 3.

Mutation of NRG1 and NRG2 restores invasive growth in a gal83 mutant. Wild-type (GAL83) and gal83Δ strains carried nrg1 and nrg2 mutations, singly and in combination, as indicated. Cells were spotted on YEP plus 2% glucose containing 2.5% agar, incubated at 30°C for 2 days, and washed. Strains tested were MATa (MCY4700 series in Table 1). The gal83 mutants all carry the K. lactis URA3 marker. +, wild-type NRG alleles.

FIG. 4.

The gal83 and nrg mutations affect STA2-lacZ expression. Strains with the indicated genotype were transformed with pLCLG-Staf, a centromeric plasmid carrying STA2-lacZ (18). The STA2 promoter is nearly identical to the FLO11 promoter for 3.5 kb, except for two 20- and 64-bp insertions (21). (A) Strains were those shown in Fig. 1. Transformants were grown to mid-log phase in CSM plus 2% glucose lacking leucine to select for the plasmid and were shifted to CSM plus 0.05% glucose for the indicated times. β-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units, as described previously (47). Values are average activity for four transformants, and standard errors are indicated. (B) Strains were those shown in Fig. 3. Transformants were grown overnight in selective CSM plus 2% glucose, diluted to an OD₆₀₀ of 0.1 in YEP plus 2% glucose, and grown to an OD₆₀₀ of 0.5. An aliquot of cells was shifted to YEP plus 0.05% glucose for 180 min. Assays were as described for panel A.

FIG. 5.

Microscopic examination of mutants. Cells were spread onto CSM lacking glucose (A) or CSM plus 0.1% glucose (B) and incubated for 16 to 24 h at 26°C to form microcolonies as described in Materials and Methods. Strains were those shown in Fig. 1 and other auxotrophically matched reg1 strains. Bars, 10 μm.

FIG. 6.

Agar invasion and filamentation of strains expressing FLO11 from the S. pombe adh⁺ promoter. (A) Wild-type and reg1 mutant strains carrying the SpADHp-FLO11 allele were spread onto CSM containing 0.1% or no glucose and allowed to form microcolonies as described in Materials and Methods. (B) Strains of the indicated genotypes were tested for invasive growth on YEP plus 2% glucose with 2.5% agar at 26°C. Plates were photographed after 10 days. The REG1 SpADHp-FLO11 strain should have a slight advantage for invasive growth because it carries the ura3 mutation; nonetheless, the reg1Δ::URA3 SpADHp-FLO11 strain was more invasive. (C) Strains of the indicated genotypes were examined as described for panel A. Bars, 10 μm.

FIG. 7.

Model for roles of the Snf1 kinase in invasive growth. The Snf1-Gal83 form of the kinase regulates FLO11 and possibly other Nrg-repressed genes that affect adhesion. Snf1 may also affect FLO11 expression by other mechanisms. Snf1-Gal83 and Snf1-Sip2 affect filamentation by a pathway(s) that does not involve FLO11 or Nrg repressors. (A) These two kinase forms may function redundantly in the same pathway. (B) Alternatively, the two kinases may function in two distinct pathways, either of which suffices for filamentation. Moreover, the role of Snf1 in filamentation may involve multiple targets. Snf1-Sip1 also has an inhibitory effect on invasive growth, but the mechanism is not yet understood.