Award details

The role of sterol - and sphingolipid - rich lipid domains in plant plasma membrane protein trafficking.

Reference	BB/C507561/1
Principal Investigator / Supervisor	Professor Paul Dupree
Co-Investigators / Co-Supervisors	Professor Kathryn Lilley, Dr Thilo Weimar
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	303,840
Status	Completed
Type	Research Grant
Start date	01/07/2005
End date	30/06/2008
Duration	36 months

Abstract

The aim of the proposed project is to investigate the hypothesis that specific membrane lipids or lipid domains (lipid rafts) are involved in trafficking of distinct classes of plant cell surface proteins. The lipid domain phenomenon might regulate both trafficking and activity of a large number of plant cell surface proteins such as metabolite transporters and proteins involved in development and stress response signalling. Although the phenomenon has had a huge impact on mammalian cell biology, it has scarcely been studied in plants, in part because of the absence of experimental tools and lipid domain markers. We have recently obtained strong evidence for detergent-resistant membranes (DRMs) that are enriched in phytosterols, phytosphingolipids and specific plasma membrane proteins, supporting the lipid domain hypothesis. The proteins in these DRMs include plasma membrane ATPases, MDR transporters, certain GPI-anchored proteins, and probably also lipid-modified proteins at the cytosolic surface of the domains. We now propose to identify comprehensively the integral membrane proteins in these DRMs. To control for specificity in detergent resistance, we will use quantitative proteomics techniques of isotopic labelling (ICAT) coupled with 2D LC-MS/MS (MudPIT). We also propose to identify comprehensively integral plasma membrane proteins by non-gel based proteomics, using the same quantitative proteomics ICAT technique to identify plasma membrane preparation contaminants. We will thus be able to determine which plasma membrane proteins are localised to the DRMs, and identify those that are apparently excluded from these domains. The subcellular localisation of selected DRM and non-DRM markers will then be determined in transgenic plants, to reveal any differences in localisation. We will next test the involvement of sterols and sphingolipids in membrane trafficking of proteins found in the DRMs. Using mutants in sterol or in sphingolipid synthesis, alteration in trafficking of DRM proteins will be assessed by microscopy of GFP fusions of selected proteins, and also by comparison of DRM composition between mutant and wild type. This combined proteomic and cell biological study will give a novel insight into the plant cell processes connected to membrane lipid domains. It will provide markers for visualisation and studies of these lipid-dependent processes, and thus open up a new field in trafficking in plants.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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