Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing

As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predicti ons for about half of the cDNAs sequenced, we have developed and tested a s trategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate Nand C-terminal gr een fluorescent protein fusions of cDNAs to examine the intracellular local izations of >100 expressed fusion proteins in living cells. The entire anal ysis is suitable for automation, which will be important for scaling up thr oughput. For >80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the locali zation was able to support these predictions in 75% of cases. For those cDN As where no homologies could be predicted, the localization data represent the first information.