University of Groningen Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins Kralt,

Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. INTRODUCTION The gateway between the cytoplasm and the nuclear interior is formed by the nuclear envelope (NE)–embedded nuclear pore complex (NPC) through which bidirectional transport between the two compartments occurs. Small solutes and proteins readily diffuse through the NPC, whereas diffusion of larger macro­ molecular complexes is slow or prevented. The presence of a nuclear localization signal (NLS) or nuclear export signal recog­ nized by transport receptors permits transport using an energy­ dependent mechanism (Chook and Suel, 2011) and also allows the passage of larger molecules. Specific interactions of these transport receptors with phenylalanine­glycine repeats of nu­ cleoporins (Nups) filling the central channel of the NPC mediate the transport of soluble cargoes (Fiserova et al., 2010; Peleg and Lim, 2010; Yang, 2013), and receptor–cargo binding and release are dictated by a gradient of RanGTP across the NPC (Kalab et al., 2002; Fried and Kutay, 2003; Madrid and Weis, 2006; Cook et al., 2007). Monitoring Editor Karsten Weis ETH Zurich Received: Mar 31, 2015 Revised: Jul 7, 2015 Accepted: Jul 8, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E15-03-0184) on July 15, 2015. L.M. V. and A.K. initiated the project and wrote the manuscript together with all of the other authors. A.K. performed all experiments except the biochemical and structural studies in Figure 2, which were performed and analyzed by R.K. L. and G.C. The experiments in HEK293T cells in Figure 6, B and C, and Supplemental Figure S2 were designed by A.K. , M.F. , and N.J.B. , performed by A.K. and N.J.B. , and analyzed by A.K. and M.F. Experiments in Figures 5 and 6A were performed with the help of V.v. d.B. ; A.S. performed experiments in Figure 4. Address correspondence to: Liesbeth M. Veenhoff (l.m.veenhoff@rug.nl). © 2015 Kralt 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: DAM, DNA adenine methyltransferase; ER, endoplasmic reticulum; FKBP, FK506-binding protein; FL, full length; FRB, FKBP12-rapamycin binding domain; GFP, green fluorescent protein; IBB, importin β binding; INM, inner nuclear membrane; NE, nuclear envelope; NLS, nuclear localization signal; NPC, nuclear pore complex; Nup, nucleoporin; ONM, outer nuclear membrane. Annemarie Kralta, Noorjahan B. Jagalurb, Vincent van den Boomc, Ravi K. Lokareddyd, Anton Steena, Gino Cingolanid, Maarten Fornerodb, and Liesbeth M. Veenhoffa aEuropean Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, Netherlands; bDepartments of Biochemistry and Pediatric Oncology, Erasmus MC/Sophia, 3015 CN Rotterdam, Netherlands; cDepartment of Experimental Hematology, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, Netherlands; dDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107 http://www.molbiolcell.org/content/suppl/2015/07/13/mbc.E15-03-0184v1.DC1. html Supplemental Material can be found at: