BioMatNet Item: QLK3-2001-00533 - Engineering yeast for efficient expression of heterologous membrane transporters (EFFEXPORT)

[BioMatNet Database - FP5 Quality of Life Programme]

QLK3-2001-00533
Engineering yeast for efficient expression of heterologous membrane transporters (EFFEXPORT)

Contacts

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Quality of Life - 3. The Cell Factory

Type of Project	Shared cost research
Contract No	QLK3-2001-00533
Total Cost
EC Contribution	2,490,400 EUR
Start Date	01-01-2002
Duration	36 Months

Abstract

Expression of human proteins in a readily usable heterologous system is a powerful mean to study their function and structure and to discover novel targets and therapeutic routes directly associated with the causes of disease. Common yeast is ideal to adapt this technology to the human genome scale. Yet many laboratories have the same fundamental problem: the failure of many membrane proteins (the main target of pharmaceutical products) to be functionally expressed in yeast due to incorrect delivery or maintenance at the plasma membrane. The EFFEXPORT project aims to engineer yeast into a more reliable system for expression of heterologous membrane transporters. The novel yeast strains and associated strategies will be used to develop a new type of screening assay for identifying chemical compounds that act selectively on transporters of interest.

Objectives

The EFFEXPORT project has four main objectives:

to precisely identify processes and mechanisms in the yeast membrane trafficking apparatus that interfere with or limit the functional expression of heterologous membrane proteins.
to isolate mutations and develop expression strategies enabling several heterologous transporters of different origins to be active in yeast when normally they are not.
to combine these solutions and to construct a host strain as universal as possible for reliable and stable expression of foreign membrane transporters.
to use this improved expression system to develop several yeast-based assays for high-throughput screening for novel therapeutics acting on a selection of cell-surface transporters of high medical interest.

Activities

The work will begin with the application of a battery of biochemical and cell biology assays aimed at better understanding why so many heterologous membrane transporters are not functional when expressed in yeast. Cell responses by yeast strains expressing these heterologous proteins will also be examined by means of genome-wide transcriptional analysis. Several genetic approaches will then be applied in parallel to a selection of transporters in order to improve their functional expression in yeast. On the basis of recent data obtained independently by several project partners, the isolation of mutations in the transgenes and of others in the yeast chromosomes is anticipated.

Novel strategies for overexpressing the heterologous transporters in a stable manner will also be established. On the basis of the data gathered from the parallel analysis of plant, animal and human membrane transporters, a novel yeast host strain and associated expression strategies will then be designed, constructed and tested for their ability to promote functional expression of many different heterologous transporters. From this novel material will be isolated a complete collection of mutants defective in all yeast transport functions characterised to date. This collection will be an invaluable tool, notably for the systematic functional analysis of the great many human genes encoding putative transporters uncovered by the genome project. Finally, assays based on growth tests and on cell responses to compound transport will be developed, standardised and converted into formats allowing automation and miniaturisation.

Deliverables

The deliverables of EFFEXPORT include:

a set of yeast strains expressing heterologous membrane transporters (native, GFP- or HA-tagged) of pharmaceutical or agro-industrial interest.
data of the detailed analysis of the subcellular location, stability and post-translational modifications of each heterologue.
global data of genome-wide transcription profile analysis of cells expressing the heterologes.
a list of cis- and trans-acting mutations and trans-acting factors improving the functional expression of the heterologous transporters in yeast.
an universal host (UH), i.e. yeast strains and associated strategies for effective expression of heterologous membrane proteins in yeast.
a complete collection of transport mutants isolated in the UH.
multiple miniaturized assays for high throughput screening (HTS) of new therapeutics acting on a selection of human transporters.

Contacts

Coordinator

Bruno ANDRÉ / Université Libre de Bruxelles IBMM - Physiologie Cellulaire - CP300

Participant