Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth - PubMed (original) (raw)

Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth

A F Roth et al. Mol Cell Biol. 2000 Dec.

Abstract

The inappropriate expression of the a-factor pheromone receptor (Ste3p) in the MATa cell leads to a striking inhibition of the yeast pheromone response, the result of a functional interaction between Ste3p and some MATa-specific protein. The present work identifies this protein as Asg7p. Normally, expression of Ste3p and Asg7p is limited to distinct haploid mating types, Ste3p to MATalpha cells and Asg7p to MATa cells. Artificial coexpression of the two in the same cell, either a or alpha, leads to dramatic inhibition of the pheromone response. Ste3p-Asg7p coexpression also perturbs the membrane trafficking of Ste3p: Ste3p turnover is slowed, a result of an Asg7p-mediated retardation of the secretory delivery of the newly synthesized receptor to the plasma membrane. However, in the absence of ectopic Ste3p expression, the asg7Delta mutation is without consequence either for pheromone signaling or overall mating efficiency of a cells. Indeed, the sole phenotype that can be assigned to MATa asg7Delta cells is observed following zygotic fusion to its alpha mating partner. Though formed at wild-type efficiency, zygotes from these pairings are morphologically abnormal. The pattern of growth is deranged: emergence of the first mitotic bud is delayed, and, in its place, growth is apparently diverted into a novel structure superficially resembling the polarized mating projection characteristic of haploid cells responding to pheromone. Together these results suggest a mechanism in which, following the zygotic fusion event, Ste3p and Asg7p gain access to one another and together act to repress the pheromone response, promoting the transition of the new diploid cell to vegetative growth.

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Figures

FIG. 1

FIG. 1

Slowed Ste3p turnover in MATa cells responding to pheromone. Turnover of Ste3p was monitored in three isogenic yeast strains carrying a GAL1-STE3 construct in place of the wild-type STE3 locus: the wild-type _MAT_α strain NDY1132, the wild-type MATa strain NDY1140, and the mfa1Δ mfa2Δ MATa strain SY2555. In addition, strains were transformed by pND541, a plasmid which expresses an HA-tagged Ura3p from the GAL1 promoter. A 3-h period of Ste3p and HA-Ura3p expression was induced from cultures growing in raffinose medium with the addition of galactose and subsequent termination by glucose addition. One of two SY2555 cultures was treated with two doses of α-factor: a dose of 6 × 10−6 M administered 30 min prior to glucose addition and a dose of 15 × 10−5 M administered 30 min after the glucose addition. Protein extracts were prepared from culture aliquots, removed at the time of glucose addition (0-h time point) and at indicated times thereafter, and subjected to Western analysis both with Ste3p-specific antibodies (top) and with the HA.11 monoclonal antibody (bottom). The HA-Ura3p control provides the experiment with an internal standard of a protein not subject to rapid turnover.

FIG. 2

FIG. 2

ASG7 is the a-specific gene responsible for the Daf phenotype. Approximately 300 cells were spotted onto rich YP plates containing either 2% glucose or 2% galactose and allowed to grow for 2 days at 30°C. Cells from eight isogenic strains SY2150, SY2560, SY2561, NDY1050, and NDY1077 (see Table 1 for genotypes), as well as SY2560, NDY1050, and NDY1078, transformed by pND997 (p_ADH1-ASG7_), a plasmid construct which constitutively expresses ASG7 from the ADH1 promoter, were tested. The relevant features of the strain genotypes are indicated to the left of growth spots.

FIG. 3

FIG. 3

Ste3p and Asg7p coexpression impairs the response to α-factor. β-Galactosidase activity from a FUS1-LacZ reporter construct was used as a measure of the α-factor-induced transcriptional response in six isogenic FUS1-LacZ bar1Δ STE2+ MATa strains: NDY1123, NDY1124, NDY1131, NDY1172, NDY1173, and NDY1174 (see Table 1 for strain genotypes). Cultures growing in YP-galactose (2%) medium were treated for 1 h with 10−9, 10−8, 10−7, 10−6, or 10−5 M α-factor or were mock treated in parallel with no pheromone. Dose-response curves for induced β-galactosidase activity versus α-factor concentration are shown. As indicated to the right of each of the plots, the six strains differ both at the STE3 locus, which is _ste3_Δ or GAL1-STE3, and at the ASG7 locus, which is wild-type ASG7, _asg7_Δ, or ADH1-ASG7.

FIG. 4

FIG. 4

Asg7p is responsible for the slowed Ste3p turnover. Ste3p turnover was assessed by Western blotting as in Fig. 1; the loss of Ste3 antigen following the glucose repression of GAL1-STE3 constructs was monitored. (A) ASG7 encodes the pheromone-inducible factor responsible for slowed Ste3p turnover. The isogenic ASG7+ and asg7Δ MATa _GAL1-STE3 mfa1Δ mfa2_Δ strains SY2555 and NDY1086 were cultured and treated with α-factor or were mock treated as described above for Fig. 1. (B) Constitutively expressed ASG7 suffices to retard Ste3p turnover in a cells. The isogenic _asg7_Δ, and ADH1-ASG7 MATa _GAL1-STE3 mfa1Δ mfa2_Δ strains NDY1124 and NDY1131 were cultured as described above for Fig. 1, except α-factor treatment was omitted. (C) Ectopic ASG7 expression in α cells retards Ste3p turnover. The _GAL1-STE3 MAT_α SY2150 cells transformed by the ADH1-ASG7 plasmid pND997 or by the empty-vector plasmid (wt) were cultured as described for Fig. 1, except α-factor treatment was omitted.

FIG. 5

FIG. 5

Asg7p delays Ste3p delivery to the cell surface. A 60-min period of receptor expression from GAL1-STE3Δ365 was induced with galactose addition and terminated with the subsequent addition of glucose. Cell growth was continued for an additional 30 min, affording the newly synthesized receptor the opportunity to reach the cell surface. Culture aliquots at various time points were either treated with proteases (+) or were mock treated in parallel (−) (see Materials and Methods). Protein extracts prepared from these cells were subjected to Western analysis using Ste3p-specific antibodies. (A) Constitutive ASG7 expression delays the delivery of Ste3p to the cell surface in MATa cells. MATa _GAL1-STE3Δ365 mfa1Δ mfa2_Δ cells, either _asg7_Δ (NDY1125) or ADH1-ASG7 (NDY1141), were cultured as described above. At 30 and 60 min following initiation of the galactose-induced pulse and 30 min after the subsequent addition of glucose (the 90-min time point), culture aliquots were removed for protease shaving. Indicated at right are the positions both of the undigested Ste3Δ365p receptor and of a receptor digestion product corresponding to the protected cytoplasmic tail domain (CTD) plus the most C-terminal of the seven receptor transmembrane domains. (B) The Asg7p-mediated delay in Ste3p surface delivery occurs in a/α diploid cells which do not express the subunits of the heterotrimeric G protein. Localization of the Ste3Δ365p expressed in MATa/α and _MAT_α/α cells cultured as described above was assessed at the 90-min time point (following the 30-min glucose chase period) via the protease-shaving protocol. The MATa/α strains used, NDY1176 and NDY1178, and the _MAT_α/α strains used, NDY1185 and NDY1186, were ASG7+/ASG7+ and ADH1-ASG7/ASG7+, respectively. (Wild-type ASG7 is expected to be transcriptionally silent in both a/α and α/α contexts [24]). For brevity, the portion of the gel displaying the lower-molecular-weight CTD fragment is not shown. (C) The Asg7p-mediated delay in Ste3p surface delivery occurs in cells with the Gβ subunit-encoding gene STE4 deleted. MATα GAL1-STE3Δ365 cells (NDY1200) as well as MATα GAL1-STE3Δ365 ADH1-ASG7 (NDY1204) and _MATα GAL1-STE3Δ365 ADH1-ASG7 ste4_Δ::LEU2 cells (NDY1225) were cultured and treated with the protease-shaving protocol as described for panel B.

FIG. 6

FIG. 6

Zygotes from asg7 matings are morphologically deranged. Wild-type W303-1B _MAT_α cells were mated to either wild-type W303-1A MATa cells or the isogenic _asg7_Δ strain NDY1089 for 2.5 or 5 h. Selected zygotes visualized by Nomarski optics are shown. White arrows, new mitotic buds; black arrows, stalk-like structures from the mitotic bud often seen to emerge for asg7 zygotes.

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