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Karyopherin-Mediated Import of Integral Inner Nuclear Membrane Proteins

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Karyopherin-Mediated Import of Integral Inner Nuclear Membrane Proteins Vol 442|31 August 2006|doi:10.1038/nature05075 ARTICLES Karyopherin-mediated import of integral inner nuclear membrane proteins Megan C. King1*, C. Patrick Lusk1* &Gu¨nter Blobel1 Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble ‘classical’ nuclear localization signals. These sequences can mediate direct binding to karyopherin-a and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-a, karyopherin-b1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane. Integral membrane proteins of the INM contribute to the organiz- fluorescent protein (YFP) fusions at endogenous (Supplementary ation of lamins and chromatin at the nuclear envelope1; mutations in Fig. 2a) and elevated levels of expression (Fig. 1b and Supplementary genes encoding lamins and lamin-associated integral INM proteins Fig. 2b). This contrasts with the distribution of a typical ER protein, are tied to a clinically diverse array of genetic diseases referred to as which evenly distributes between the perinuclear ER and the cortical laminopathies2. To reach the INM, integral membrane proteins must ER (Supplementary Fig. 2c). To ensure that Heh1 and Heh2 accumu- move past nuclear pore complexes (NPCs), large protein channels late specifically in the INM, we performed immunoelectron that regulate the nucleocytoplasmic transport of soluble molecules3. microscopy. As shown in Fig. 1c, when immunoelectron microscopy Because the outer nuclear membrane (ONM) is contiguous and was performed using antibodies directed against the C-terminal YFP functionally equivalent with the endoplasmic reticulum (ER), INM moiety of Heh2–YFP, gold particles were found almost exclusively proteins might reach the cytoplasmic aspect of NPCs by diffusion. along the inside of the nuclear envelope (see Fig. 1d for higher Indeed, it has been proposed that integral INM proteins diffuse magnification). We determined that over 50% of Heh1–YFP and laterally along the pore membrane and are then selectively retained in Heh2–YFP was localized at the nuclear envelope in wild-type cells, the nucleus through interactions with elements of the nuclear and of that greater than 70% was found at the INM (Supplementary architecture4. However, as they pass along the pore membrane Fig. 2d). Thus, Heh1 and Heh2 are useful examples with which to both transmembrane segment(s) and hydrophilic domains probably examine the targeting of integral INM proteins. encounter elements of the NPC, which may need to restructure By further analysing the amino acid sequence of Heh1 and Heh2 locally to accommodate their passage. A recent study in mammalian and their human orthologues, MAN1 and LEM2, we uncovered the cells demonstrates that transport of integral membrane proteins to presence of sequences similar to nuclear localization signals (NLSs, the INM requires energy5, suggesting the presence of an active Fig. 1a; sequences in Supplementary Table 1), sequence elements that transport pathway in lieu of, or in addition to, passive diffusion/ promote active nuclear import of soluble proteins8,9. This finding retention. raised the possibility that mediators of soluble nuclear transport may contribute to INM targeting. Heh1 and Heh2 are yeast INM proteins In order to examine further the mechanism by which integral The Ran cycle is required for INM targeting membrane proteins are targeted to the INM, we searched for We first developed an inducible system for monitoring the targeting potential INM proteins in the model organism Saccharomyces of Heh1 and Heh2 as YFP fusions produced under the control of the cerevisiae. We identified two yeast INM proteins based on homology GAL1 promoter. This allowed us to monitor conditionally the fate of with the mammalian INM paralogues MAN1 and LEM2. Common newly synthesized cargo molecules in live cells. To test the hypothesis to both the yeast and human proteins are an amino-terminal region that mediators of soluble transport contribute to integral INM likely to form a helix-extension-helix (HEH) fold, two transmem- protein targeting, we examined Heh1–YFP and Heh2–YFP localiz- brane domains, and a region of homology after the second trans- ation in yeast mutants in which nuclear transport is globally inhibited membrane domain (HEH/MAN1 carboxy-terminal homology through the disruption of the GTP loading and hydrolysis of the domain, CTHD)6 (Fig. 1a). To reflect this homology, we now refer GTPase Ran. GTP-bound in the nucleus and GDP-bound in the to these genes as HEH1 (formerly called SRC1 (ref. 7)) and HEH2 cytoplasm, Ran coordinates the bidirectional transport of macro- (systematic name YDR458C). Both Heh1 and Heh2 localize exclu- molecules across the nuclear envelope8,10. Proper cycling of Ran (and sively to the nuclear envelope when produced in yeast as yellow thus nuclear transport) is disrupted at 34 8C in yeast strains carrying 1Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. *These authors contributed equally to this work. 1003 © 2006 Nature Publishing Group ARTICLES NATURE|Vol 442|31 August 2006 temperature-sensitive mutations in the Ran guanine nucleotide the cortical ER accumulation correlates with a disruption in INM exchange factor (Mtr1/Srm1/Prp20) or the Ran GTPase-activating targeting, as the percentage of Heh1–YFP at the nuclear envelope protein (Rna1). When Heh1–YFP and Heh2–YFP were produced in a dropped markedly from over 50% in wild-type cells to less than 5% in strain harbouring a mutation in RanGEF (mtr1-1)11 they localized the mtr1-1 strain at 34 8C, with essentially no label at the INM primarily to the nuclear envelope at the permissive temperature (Supplementary Fig. 3a). There was also an appreciable defect in (25 8C).Notably,ifHEH1–YFP and HEH2–YFP induction was targeting of Heh1–YFP in mtr1-1 cells at the permissive temperature preceded by shifting cells to 34 8C, Heh1–YFP and Heh2–YFP (25 8C, compare Fig. 1b, e and see Supplementary Fig. 3a). Notably, accumulated at the periphery of the cell, probably in the cortical mislocalization of Heh1–YFP and Heh2–YFP was reversible, as ER (Fig. 1e). Using immunoelectron microscopy, we confirmed that shifting of the culture back to 25 8C (under repressive conditions) led to recovery of nuclear envelope localization (Fig. 1e). Similar mislocalization of Heh1–YFP was observed in a strain harbouring a temperature-sensitive mutation in RanGAP (Rna1), and there were no changes to the distribution of Heh1–YFP in wild-type cells at 34 8C (Supplementary Fig. 3b, c). Nuclear envelope targeting requires karyopherins Ran regulates nuclear transport by modulating soluble nuclear transport factors called karyopherins8,10. The ‘classical’ nuclear import pathway is initiated when the cNLS (the ‘classical’ NLS refers to the NLS in the SV40 T-antigen12, see Supplementary Table 1) of a cargo molecule is recognized by the import receptor karyopherin-a (importin-a/Kap60/Srp1) in an interaction that is stabilized by binding of karyopherin-b1 (importin-b1/Kap95). This ternary com- plex traverses the NPC and is disassembled by binding to Ran-GTP within the nucleus. To examine whether karyopherins are involved in Heh1/Heh2 targeting, we monitored the localization of Heh1–YFP and Heh2–YFP in strains with temperature-sensitive mutations or deletions in various karyopherin genes. At the non-permissive temperature in strains harbouring mutations in either KAP60 (srp1-31)13 or KAP95 (kap95-L63A)14, Heh1–YFP and Heh2–YFP failed to accumulate exclusively at the nuclear periphery, and localized throughout the cortical ER (Fig. 2a and data not shown). We did not detect any defects in the localization of Heh1–YFP or Heh2–YFP in strains harbouring mutations or deletions in other karyopherin genes including KAP121 (kap121-34 (ref. 15), Fig. 2a), Figure 1 | Heh1–YFP and Heh2–YFP localize to the INM in a Ran-dependent manner. a, Diagram of the likely topology of the indicated proteins in the INM. Conserved domains are coloured green (HEH/LEM domain) and magenta (HEH/MAN1 CTHD); transmembrane segments are in black. The RNA recognition motif (RRM) of MAN1 is coloured grey. Cyan circles indicate putative nuclear localization signals. Numbers represent amino acid Figure 2 | Kap60 directly interacts with Heh2 and is required for nuclear residues. b, Fluorescence micrograph of Heh1–YFP in a wild-type yeast envelope targeting. a, Fluorescence images of the subcellular distribution strain (CPL160). c, Immunoelectron micrograph of Heh2–YFP in a of Heh2–YFP produced in the indicated karyopherin mutants (CPL165, wild-type yeast strain (CPL148) labelled with anti-GFP antibodies followed CPL166, CPL167) at the non-permissive temperature (34 8C). b, Schematic by 10-nm-diameter gold-particle-conjugated secondary antibody. N denotes of Heh2 with coloration as in Fig. 1a. The amino acid sequence of the Heh2 the nucleus. d, Detail of electron micrograph in c. Arrows indicate the NLS is given; numbers are amino acid residues. c, The binding of purified nuclear envelope. Scale bar in c, d, 100 nm. e, Fluorescence micrographs of recombinant Kap60 and Kap121 to GST fusions of the indicated truncations Heh1–YFP and Heh2–YFP produced in the RanGEF mutant (mtr1-1) strain of Heh2 was assessed by GST pull-down assays. Equivalent amounts of (CPL162 and CPL163) at the indicated temperature.
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