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. 2011;6(12):e28799.
doi: 10.1371/journal.pone.0028799. Epub 2011 Dec 8.

The ankyrin repeats and DHHC S-acyl transferase domain of AKR1 act independently to regulate switching from vegetative to mating states in yeast

Piers A Hemsley  1 , Claire S Grierson
Affiliations

The ankyrin repeats and DHHC S-acyl transferase domain of AKR1 act independently to regulate switching from vegetative to mating states in yeast

Piers A Hemsley et al. PLoS One. 2011.

Abstract

Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AKR1 S-acylates itself in trans and this does not require the ankyrin repeats.
S-acylation assays of AKR1 variants expressed in yeast were performed using the biotin switch method whereby hydroxylamine is used to specifically cleave S-acyl groups revealing sulfhydryls which are subsequently labelled with biotin. Samples are then passed through a neutravidin column and S-acylation state is assayed as a function of recovery by the column using antibodies against the protein of interest. Negative controls substitute Tris, which does not cleave S-acyl groups, for Hydroxylamine. +indicates presence of hydroxylamine, −indicates absence of hydroxylamine. EX –S-acylated AKR1 variants detected by the biotin switch method, LC – loading control to show the total amount of AKR1 variant (regardless of S-acylation state) in each sample. (A) AKR1 is able to covalently bind acyl groups (auto-S-acylate) and disruption of the DHHC domain by introduction of the C500S mutation abolishes this auto acylation. AKR1 ΔN maintains the ability to auto acylate and co-expression of AKR1 ΔN and AKR1 C500S leads to S-acylation of AKR1 C500S (upper band in lane marked with *). (B) S-acylation of AKR1 C500S in trans occurs in a wild type background. Expression of AKR1 C500S in WT cells (AKR1 fully functional) produces the same S-acylation of AKR1 C500S in trans as co-expression of AKR1 C500S with AKR1 ΔN in akr1Δ cells. (C) Western blot showing that the total amount of AKR1 C500S detected increases in conditions where this protein is S-acylated in trans, suggesting that S-acylation promotes AKR1 stability. Expression of AKR1 C500S in WT cells or co-expression in akr1Δ cells with AKR1 ΔN leads to higher levels of AKR1 C500S being detected than expression of AKR1 C500S alone in akr1Δ cells. LC – loading control: Histone H3.
Figure 2
Figure 2. S-acylation of over expressed GFP-YCK2 requires the DHHC domain of AKR1.
S-acylation of GFP-YCK2 is supported by AKR1 or AKR1 ΔN but not by AKR1 C500S. The presence of the AKR1 ankyrin repeats on the non-S-acyl transferase competent AKR1 C500S improves S-acylation of GFP-YCK2 when co-expressed with AKR1 ΔN. S-acylation states were assayed by the biotin switch method, +indicates presence of hydroxylamine, −indicates absence of hydroxylamine. EX – S-acylation state of GFP-YCK2, LC – loading control for GFP-YCK2.
Figure 3
Figure 3. AKR1 interacts with Gβγ through its ankyrin repeats.
(A) Over-expression of the Gβ subunit STE4 from the GAL1 promoter of pJB9 leads to cell cycle arrest and failure to grow on galactose medium (Gal). All strains grew well on glucose medium (Glu). Over expression of AKR1 or AKR1 C500S from high copy number vectors suppresses STE4 over expression induced cell cycle arrest whereas AKR1 ΔN or non-membrane associated AKR1 ankyrin repeats (AKR1 ANKS) do not. (B) Yeast-2-hybrid analysis of the interaction between AKR1 variants and the Gγ subunit STE18. AKR1 and AKR1 C500S both suppressed the ade2Δ red phenotype and produced strong growth on medium selecting for activation of HIS3 and ADE2 reporter genes as a result of interaction with STE18 whereas AKR1 ΔN did not. –LW: control medium, -LWHA: selection medium for reporter activation. +denotes pAI (positive control vector), −denotes pDL2 (negative control vector), pPR3N – empty prey vector control, STE18 – pPR3N expressing STE18. (C) Yeast-2-hybrid assay demonstrating direct interaction between the ankyrin repeats of AKR1 and the Gγ subunit STE18. The AKR1 ankyrin repeats suppress the ade2Δ red phenotype and produce strong growth on medium selecting for activation of HIS3 and ADE2 reporter genes as a result of the AKR1 ankyrin repeats interacting with STE18. Controls are the same as for part B.
Figure 4
Figure 4. AKR1 requires both the DHHC domain and ankyrin repeats for rescue of phenotypic and mating pathway activation defects, but they do not need to be on the same molecule.
(A) akr1Δ mutants show a reduction in WT morphology cells (black bars) and an increase in the proportion of cells with schmoo-like (grey bars) or multiple bud phenotypes (white bars). Introduction of AKR C500S to akr1Δ cells (akr1Δ+C500S) suppresses the schmooing phenotype but increases the number of cells showing a multiple bud phenotype. Introduction of AKR1 ΔN to akr1Δ cells (akr1Δ+ΔN) fails to suppress the schmooing phenotype but does reduce the number of cells that show a multiple bud phenotype. Co-expression of AKR1 C500S and AKR1 ΔN (akr1Δ+C500S+ΔN) in akr1Δ cells restores all morphological phenotypes to that observed for expression of full length WT AKR1 (akr1Δ+AKR1). The data represents the average of 3 independent experiments with >220 cells counted per genotype per experiment. Representative images of cultures used to generate these data are shown in Figure S3. Error bars represent 1 standard deviation. (B) akr1Δ mutants show a temperature sensitive phenotype with growth occurring at 25°C but not at 37°C. Introduction of AKR C500S (akr1Δ+C500S) or AKR1 ΔN to akr1Δ cells (akr1Δ+ΔN) fails to suppress the temperature sensitive phenotype. Co-expression of AKR1 C500S and AKR1 ΔN (akr1Δ C500S+ΔN) in akr1Δcells restores temperature resistance as effectively as full length WT AKR1 (akr1Δ AKR1). (C) akr1Δmutants show low level induction of the mating pathway in the absence of mating pheromone as measured by real time PCR of FUS1 mRNA. Introduction of AKR1 C500S to akr1Δcells suppresses FUS1 induction significantly compared to akr1Δ while AKR1 ΔN only partly suppresses FUS1 induction. Error bars represent a 95% confidence interval calculated from 3 technical replicates. Data are representative of 3 independent experiments.
Figure 5
Figure 5. Speculative model of AKR1 function suggested by our results and the literature.
Our results are consistent with a model where the ankyrin repeats suppress aspects of Gβγ-induced mating pathway activation independently of the DHHC domain. In the absence of mating pheromone the ankyrin repeats suppress basal Gβγ signalling through the STE20 MAPK pathway, possibly by directly binding (see results in Figure 3) and sequestering Gβγ or a Gβγ containing complex released by basal STE3 activity away from downstream mating pathway components The DHHC domain suppresses inappropriate mating pathway activation by ensuring STE3 levels are kept low via the S-acylation of YCK2, which in turn would phosphorylate STE3 at the plasma membrane leading to STE3 endocytosis. These processes would both help to maintain low levels of basal Gβγ signalling and promote vegetative growth. This model suggests the following explanations for the phenotypes that we have observed. The low level mating pathway activation in AKR1ΔN expressing cells could be due to free Gβγ released by basal STE3 activity partially activating the mating pathway in the absence of AKR1 ankyrin repeats (which would otherwise suppress Gβγ activity). AKR1 C500S, unable to S-acylate YCK2, would lead to a failure in constitutive endocytosis of STE3 , , and the resultant elevated levels of STE3 would lead to elevated basal Gβγ release , exceeding the buffering potential of the ankyrin repeats. This would explain the slightly poorer performance of AKR1 C500S in suppressing FUS1 induction (Figure 4C). Domains of AKR1 and proteins with roles predominantly in vegetative growth are represented in white while those with roles predominantly in mating are represented in grey. Broken lines represent S-acylation activity of AKR1. Arrowed lines represent positive effects on function, barred lines represent negative effects on function. AKR1 DHHC – DHHC domain of AKR1, AKR1 ANK – ankyrin repeats of AKR1.
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