Imaging of single-molecule translocation through nuclear pore complexes

doi:10.1073/pnas.0403675101

Imaging of single-molecule translocation through nuclear pore complexes

^*Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, 1114 TAMU, College Station, TX 77843; and ^†Department of Biochemistry, Brandeis University, MS 009, P.O. Box 549110, Waltham, MA 02454

Edited by Michael Rosbash, Brandeis University, Waltham, MA, and approved July 14, 2004 (received for review May 24, 2004)

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.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: smusser{at}tamu.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; Nup, nucleoporin; FG, phenylalanine–glycine; IC, import complex; SMF, single-molecule fluorescence; NLS, nuclear localization sequence; Im α/β, importin α/β; WGA, wheat germ agglutinin; CCD, charge-coupled device; S/N, signal-to-noise.