Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School and the Dana-Farber Cancer Institute
44 Binney Street
Boston, MA 02115 U.S.A.
presenter: Marc Damelin
Marc Damelin and Pamela A. Silver
Department of Biological Chemistry and Molecular Pharmacology; Harvard Medical School and the Dana-Farber Cancer Institute; Boston, MA 02115
Rapid progress in genome sequencing has made feasible large-scale screening of protein- protein interactions. We have developed an assay based on fluorescence resonance energy transfer (FRET) to map dynamic protein interactions in living cells. DNA encoding either cyan or yellow fluorescent protein (CFP, YFP) is integrated into the genome at the locus of a target gene. The endogenous copy of the gene is thus replaced by the tagged version, and the resulting fusion is expressed off the genome. The expression and functionality of all tagged proteins are confirmed. Living cells co-expressing CFP and YFP fusions are examined under an epifluorescence microscope. Digitized images captured with a charge-coupled device (CCD) camera are analyzed quantitatively to determine significant FRET signals.
Using this approach, we have investigated the mechanism of nucleocytoplasmic transport in the model organism Saccharomyces cerevisiae. In all eukaryotic cells, cargo to be transported between the nucleus and cytoplasm is carried by a transport receptor through the nuclear pore complex (NPC). The yeast NPC is composed of 30 proteins (nups) in multiple copies and has a mass of ~60MDa. We have studied two aspects of the mechanism of NPC translocation with the FRET assay. First, we screened for interactions between two transport receptors and nups, and found that these transport receptors have overlapping pathways through the NPC. The distinct receptor-nup contacts in each pathway may serve as a receptor-specific regulatory mechanism. Second, we screened for nup-nup interactions using a panel of 15 nups, and found 11 interactions. These nup-nup interactions have led us to propose a more refined structural model of the 60MDa NPC.
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