‘Green Genes’ - Systematic subcellular localisation of novel proteins identified by large-scale sequencing identifies new regulators of membrane transport

Jeremy C. Simpson
Cell Biology and Biophysics
EMBL-Heidelberg
Meyerhofstrasse 1
69117 Heidelberg
Germany
telephone: 49 6221 387232
fax: 49 6221 387306
email: simpson@embl-heidelberg.de
prestype: Platform
presenter: Jeremy C. Simpson

Jeremy C. Simpson, Veronika E. Neubrand, Stefan Wiemann and Rainer Pepperkok

Large-scale cDNA sequencing projects are providing us with vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterisation we have tagged the first 200 of these with the green fluorescent protein (GFP), and examined the subcellular localisations of these fusion proteins in living cells. The cloning of ORFs on this scale has been made possible using a novel recombination-based cloning technology which can generate both the N-terminal and C-terminal fusions in a single reaction. Subcellular localisations to discrete compartments were determined for over 75% of the fusions, providing the first functional information about these proteins, which is of particular importance for those molecules where bioinformatic tools were unable to provide any information. The tools generated by such an approach combined with the ease of cloning, mean that automation of more functional assays for such a large set of molecules is now a reality.

Of the many molecules which we have identified which localise to organelles of the secretory pathway (endoplasmic reticulum, Golgi, endosomes, plasma membrane), we have identified a novel regulator of sorting at the level of the trans-Golgi network (TGN). This protein appears to interact with adaptor molecules, specifically regulating the export of passenger molecules en route to the plasma membrane. The overexpression of this protein acts as a potent inhibitor of membrane transport from this organelle. This novel approach to finding new molecules involved in membrane transport already appears to be fruitful.