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* ¤ , Claire S. Grierson School of Biological Sciences, University of Bristol, Bristol, United Kingdom 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 Gbc 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 Gbc regulated pathways. Citation: Hemsley PA, Grierson CS (2011) The Ankyrin Repeats and DHHC S-acyl Transferase Domain of AKR1 Act Independently to Regulate Switching from Vegetative to Mating States in Yeast. PLoS ONE 6(12): e28799. doi:10.1371/journal.pone.0028799 Editor: Ruben Claudio Aguilar, Purdue University, United States of America Received June 10, 2011; Accepted November 15, 2011; Published December 8, 2011 Copyright: ß 2011 Hemsley, Grierson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in the UK by grant number BB/D013585/1 from the Biotechnology and Biological Sciences Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: piers.hemsley@bristol.ac.uk ¤ Current address: School of Biological and Biomedical Sciences, University of Durham, Durham, County Durham, United Kingdom Introduction Heterotrimeric G-protein signalling pathways are found throughout eukaryotes and are involved in a wide range of signal transduction events. Heterotrimeric G-proteins, comprising Ga, Gb and Gc subunits, are activated by ligand bound G-protein coupled receptors (GPCRs). This generally leads to dissociation of Ga from the Gbc dimer, both of which are involved in distinct signalling activities. Little is known about which proteins link signalling from Gbc dimers to downstream effectors. The mating pheromone response of Saccharomyces cerevisiae is one of the best characterised GPCR pathways. Many proteins affecting responses to mating pheromone have been identified and characterised resulting in a broad knowledge base that is useful for further dissection of GPCR pathways. AKR1 has long been known to be a negative regulator of the mating pathway in S. cerevisiae yet the mode of suppression is unknown. The gross phenotypic defects of akr1D mutants have been proposed to be a result of simultaneous activation of both vegetative and mating pathways [1]. akr1D cells are defective for endocytosis of the a- pheromone GPCR STE3 [2] as a result of YCK2 mis-localisation [3] and show up-regulation of the STE20/STE11/STE7/FUS3 MAPK mating pathway due to increased Gbc activity [1] leading to partial cell cycle arrest and activation of mating pathway morphogenesis genes in the absence of mating pheromone. These defects result in an abnormal phenotype during vegetative growth where cells frequently form multiple buds, have over-elongated buds and often fail to complete cytokinesis resulting in a multi- nucleate cell mass with pseudohyphal characteristics. akr1D cells are not however impaired in mating efficiency or up regulation of the mating pathway in response to pheromone [1,4]. These data demonstrate that AKR1 is responsible for suppressing basal Gbc mediated mating pathway signalling in the absence of mating pheromone, but does not prevent full mating pathway activation once pheromone is detected [4]. AKR1 has more recently been demonstrated to be a Protein S- acyl transferase (PAT), also known as a palmitoyl transferase, with a diverse range of substrates including the casein kinases YCK1 and 2 [5], LCB4 [6], MEH1, SNA4, LSB6 as well as 3 proteins of unknown function [7]. AKR1 is responsible for the addition of lipid groups through thioester linkages (S-acylation) to promote or increase the membrane association of the target protein [5]. YCK2 is anchored to the membrane by two S-acyl groups where it aids in many cellular processes, including septin organisation to maintain correct cell shape [8,9,10], and phosphorylates both STE3 [3] and the FUR4 uracil permease [11] to promote their endocytosis. The defects in YCK2 S-acylation and STE3 endocytosis are thought to be major contributors to the akr1D phenotype [5,10]. AKR1 is an integral membrane protein and contains 6 ankyrin repeats and a DHHC PAT domain [12], although in AKR1 the PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28799