Imaging of single-molecule translocation through nuclear pore complexes

被引:205
作者
Yang, WD
Gelles, J
Musser, SM
机构
[1] Texas A&M Univ, Syst Hlth Sci Ctr, Dept Med Biochem & Genet, College Stn, TX 77843 USA
[2] Brandeis Univ, Dept Biochem, Waltham, MA 02454 USA
关键词
D O I
10.1073/pnas.0403675101
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 approximate to10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximate to10 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.
引用
收藏
页码:12887 / 12892
页数:6
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