Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP. INTRODUCTION Macromolecular transport across the nuclear envelope is tightly regulated by nuclear pore complexes (NPCs), which are large protein complexes embedded in the envelope (Ohno et al., 1998; Fahrenkrog et al., 2001; Rout and Aitchison, 2001; Vasu and Forbes, 2001). The central channel of each NPC is filled with its subunits (nucleoporins [Nups]), which are rich in hydrophobic amino acids, and thus it functions as a hydrophobic size-selective barrier (Ribbeck and Gorlich, 2002; Wente and Rout, 2010). These features, as well as the crowded environment of the NPCs, prevent an unregulated, free exchange of macromolecules between the cytoplasm and the nucleoplasm (Timney et al., 2006; Yang and Musser, 2006; Tetenbaum-Novatt et al., 2012). Small and hydrophobic molecules, with a maximum size in the range of 20–40 kDa, are able to pass through the NPC by passive diffusion, but large and hydrophilic molecules cannot; their passage usually requires transport mediators (Paine et al., 1975; Gorlich and Kutay, 1999; Pante and Kann, 2002; Peters, 2009). Karyopherins, which are arguably the most widely studied nuclear transport mediators, facilitate the transport of a vast array of cellular proteins/molecules (Cingolani et al., 1999, 2002; Strom and Weis, 2001; Lee et al., 2003; Cansizoghu et al., 2007). In spite of their large molecular sizes (∼100 kDa), karyopherins can pass through the nuclear pore by themselves and also together with their specific cargoes. This ability of karyopherins is mostly attributed to their unique structure, which is characterized by the presence of several repeats of the HEAT motif, which comprises a pair of amphiphilic α-helices. Karyopherins and other HEAT-rich proteins have been demonstrated to undergo flexible conformational changes that enable them to pass through the hydrophobic milieu of the pore (Kumeta et al., 2012; Yoshimura et al., 2014). Although the HEAT repeat is suitable for overcoming the hydrophobic barrier, the question still remains whether karyopherins have intrinsic directionality through the pore (Chook and Blobel, 1999). During translocation across the NPC, karyopherins interact with a number of Nups, mainly via transient hydrophobic interactions (Rout et al., 2000; Frey et al., 2006; Frey and Gorlich, 2007; Lim et al., 2006; Patel et al., 2007; Hutten et al., 2008). In vitro measurement of Monitoring Editor Karsten Weis ETH Zurich Received: Aug 28, 2015 Revised: Oct 22, 2015 Accepted: Oct 27, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E15-08-0601) on November 4, 2015. Address correspondence to: S. H. Yoshimura (yoshimura@lif.kyoto-u.ac.jp). © 2016 Lolodi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; NLS, nuclear localization signal; NPC, nuclear pore complex; Nup, nucleoporin; SPR, surface plasmon resonance. http://www.molbiolcell.org/content/suppl/2015/11/02/mbc.E15-08-0601v1.DC1 Supplemental Material can be found at: