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

Abstract

As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. 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 localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

Fig. 1. Strategy for rapid systematic localization and functional characterization of proteins encoded by novel cDNAs. Individual full coding cDNAs were PCR amplified using automatically designed primers, which also added the recombination sequences attB1 and attB2 at the 5′ and 3′ ends, respectively. These products were then recombined into the entry clones, which then served as a universal source of material for all expression vectors. N- and C-terminal GFP (CFP/YFP) fusion expression vectors were both generated from the entry clone in a single recombination reaction. These clones were then transfected into cells and the localizations of the fusion proteins recorded. This information was then combined with the bioinformatic data generated from sequence analysis and additional immunostainings using compartment-specific antibodies when appropriate.

Fig. 2. cDNA–GFP fusions express and localize to a wide variety of intracellular compartments. Vero cells were transfected with each cDNA–GFP in turn, and allowed to express the proteins for the times stated in Methods. The cells were imaged live and the localizations recorded. The figure shows examples of twelve of the localizations observed, with arrows used to point out particular structures as appropriate. Numbers indicate the percentage of individual cDNA molecules that express and localize in the categories shown. Other categories not depicted are Golgi and plasma membrane localization (6%), other unknown localizations (8%) and no expression (1%). Bar, 10 µm.