Modelling ion homeostasis in the yeast Saccharomyces cerevisiae (TRANSLUCENT-2)

The ongoing SysMo2 project TRANSLUCENT-2, is a logical continuation of the work initiated in Translucent and actively ongoing at present. The current achievements of the project have paved the way for a highly ambitious program with 4 major contributions: 1.- Standardization of reagents and methods, as well as generation of new reagents (dozens of isogenic strains lacking single and all combinations of diverse cell membrane or subcellular cation transporters and regulators such as 14-3-3 proteins; new K+-free standard medium). 2.- Set-up existing in-house technologies and implementation of new approaches to solve specific problems connected to the necessary time resolved data acquisition (real-time flux measurements, software for flux, growth rate and solid growth image analysis) 3.- Generation of new initial models (biophysical model of potassium homeostasis, stochastic spatio-temporal model) allowing predictions that can be further tested by experimental approaches, leading to more refined models. 4.- Implementation of a powerful and versatile data management system for data storage and exchange with the potential to be offered as open source platform for SysMo2 users. The aim of the present project is to capitalize on all the tools, experience and previous investment in Translucent to further expand and refine modelling of cation homeostasis including not only potassium, but other cations such as sodium, as well as intracellular compartments (vacuole, nucleus, mitochondria).

1.Electrophysiological data is required in order to refine the current model. These transport proteins include Kha1, Nha1 and Ena1. In order to isolate these channels for electrophysiological characterization they will be expressed in heterologous expression systems such as COS-7 or HEK cells. This is feasible since most yeast plasma membrane channels can be expressed in the plasma membrane of HEK cells or in Xenopus oocytes. Subsequently the current/voltage relationships and ion selectivity of the channels will be ascertained. 2.An unknown question is the sequestration of ions into the vacuole. To date there is no direct data on these currents and only indirect evidence that potassium and protons are stored in the vacuole. Of particular interest are the electrophysiological characteristics of the Nhx1 and Vnx1 transporters. Our experience is that yeast vacuolar channels do not express in mammalian expression systems or Xenopus oocytes. However these channels could be examined in isolation using combined KOs of Vnx1, Nhx1 and YVC1 and patch clamping of the yeast vacuolar membrane. 3. There is evidence that Chloride is stored in the vacuole, but to date the nature of this current has not been ascertained. However it is known that a chloride channel homolog is expressed in the vacuolar and plasma membrane (GEF1) of yeast. Therefore we propose to analyse the vacuolar membranes for chloride channels in wild-type and GEF1 knockout strains using a whole vacuolar patch clamp configuration. We have shown that GEF1 can be expressed in mammalian cells so we intend to isolate this current to determine its current/voltage relationship by heterologous expression.

IMPACT SUMMARY Impact statement I will ensure that the direct consequences of my research have as wide an impact on the public sector, educational opportunities, commercial private sector and the wider public in general as possible. Since it is not possible to predict the wider impact and implications of my research I intend to review every month any potential impact this project may have by making use of the following BBSRC diagram: found at www.bbsrc.ac.uk/science/impact/index.html Using this diagram as a basis I intend to formulate an impact action plan in order that wider impact can be achieved. This will build on the impact already anticipated listed below. At this moment we already have in mind the following initiatives: 1.- To expand the scope of our biannual meetings by including one or two general lectures from our senior members introducing the participating students and colleagues to general or advanced aspects of our specific research fields. 2.- Promote the participation of young Translucent researchers in the biannual modelling courses held at Biophysics in Humboldt University (P2). These courses are primarily for undergraduate students, but as suggested by P2 experience, they are very helpful for interested non-modellers at all stages. 3.- Similarly, the Spanish Systems Biology Network organized a course on Systems Biology (June 2009) which members of P1 and P4 attended. This will be repeated during the next years and extended to members of other partners. 4.- Recent RAE-derived funding at London Metropolitan University (P6) will allow, if our proposal is accepted, to support training of a PhD student in patch clamp techniques, yeast biology and systems biology. Impact of dissemination and/ or exploitation of results and management of intellectual property at the international level Dissemination of scientific results and achievements of the consortium is among the major objectives of our proposal. Ensuring wide and appropriate dissemination is the best way to fully exploit all project results. In order to meet this goal, the dissemination plan designed within our previous Translucent project will be refined based on our previous experience. Besides the usual academic routes for dissemination (presentations at meetings, workshops and conferences, peer-reviewed scientific primary publications), emphasis will be given to: 1) Publication of scientific reviews on the project topic. This has been already initiated in the ongoing Translucent project, as leaders of P1, 4 and 7 are preparing a review for the highly reputed publication Microbiology and Molecular Biology Reviews. 2) Planning a small workshop/conference open to other scientist in the field, by seeking independent/parallel funding (i.e COST actions, etc.). 3) Stressing the relevance of Systems Biology at the level of each institution (University, Research Center, Funding Agencies, etc.) 4) Translating our work to the non-specialist public, through general dissemination activities (e. g. project-related flyers as PDF on the WEB site or general science literature). 5) The possibility of exploitation of results derived from our work (i.e. generation of commercially interesting strains, newly designed growth media, etc.). Impact on public sector, commercial private sector and wider public in general Importantly, since we aim to define the major traits for cation homeostasis in yeast from a systems biology approach, thus gaining knowledge about the functioning of the entire organism, and given the functional conservation of these mechanisms in higher plants, our studies may pave the way for future applications in the generation of salt-tolerant crops and, therefore, have an important secondary impact in plant biotechnology. Similarly, our advances may have repercussion in human health, as certain human diseases are caused by alterations of ionic balance.