Imaging of single-molecule translocation through nuclear pore complexes
Abstract
Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 ± 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least ≈103 molecules per NPC per s, NPCs must be capable of transporting at least ≈10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.
- Publication:
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Proceedings of the National Academy of Science
- Pub Date:
- August 2004
- DOI:
- 10.1073/pnas.0403675101
- Bibcode:
- 2004PNAS..10112887Y
- Keywords:
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- BIOPHYSICS