Current Project(s) :

Systematic subcellular localisation and characterisation of novel proteins involved in membrane transport.

Membrane transport serves to ensure that proteins and lipids are routed to their site of activity. In order to do this, a wide range of machinery and accessory molecules are involved, many of which have not yet been identified. As part of one approach to resolve this we have been systematically tagging novel human cDNAs identified by the German cDNA sequencing consortium with the green fluorescent protein (GFP). These are individually transfected into cells, and the subcellular localisations of the protein products recorded (1). The discrete compartmentalisation of the eukaryotic cell means that such a visual screening approach can give valuable insight into the function of a novel protein. To date we have screened and localised over 1000 novel proteins (http://gfp-cdna.embl.de), thereby providing the first functional information about these new proteins, and making significant progress towards localising the entire human proteome (2).

The screen so far has identified over 200 proteins that localise to compartments of the secretory pathway (endoplasmic reticulum, Golgi complex, and endosomes). These are assayed for their effect on membrane transport and interesting candidates taken for further analysis and individual characterisation. For example this systematic approach has allowed us to identify a novel protein of late secretory pathway that is involved in regulation of the AP-1 protein complex (3).

The ability to down-regulate target proteins in mammalian cells now provides an excellent tool to complement overexpression assays. In this respect we have developed a platform with which to carry out large scale RNAi screens to identify more factors associated with membrane traffic pathways (4).

In collaboration with Arwyn Jones (University of Wales, Cardiff, UK) I have also been characterising candidates involved in endocytic pathways. Recently we have identified the small GTPase Rab21 as a key regulator of early endocytic events (5). Eventually it should be possible to identify all the regulators of membrane traffic – both secretory and endocytic – thereby providing a picture of how these proteins orchestrate together to ensure that that cell structure and function is maintained.

(1) Simpson JC, Wellenreuther R, Poustka A, Pepperkok R & Wiemann S (2000). Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Rep. 1, 287-292. PubMed

(2) Simpson JC & Pepperkok R (2003). Localizing the proteome. Review Article. Genome Biology 4 (12), 240.1-240.4. PubMed

(3) Neubrand VE, Will RD, Mobius W, Poustka A, Wiemann S, Schu P, Dotti CG, Pepperkok R & Simpson JC (2005). g-BAR, a novel AP-1 interacting protein involved in post-Golgi trafficking. EMBO J. 24, 1122-1133. PubMed

(4) Simpson JC, Cetin C, Erfle H, Joggerst B, Liebel U, Ellenberg J & Pepperkok R (2007). An RNAi screening platform to identify secretion machinery in mammalian cells. J. Biotechnology 129, 352-365. PubMed

(5) Simpson JC, Griffiths G, Wessling-Resnick M, Fransen JAM, Bennett H & Jones AT (2004). A role for the small GTPase Rab21 in the early endocytic pathway. J. Cell Sci. 117, 6297-6311. PubMed