Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Lem2 is essential for cardiac development by maintaining nuclear integrity.

AIMS: Nuclear envelope integrity is essential for the compartmentalization of the nucleus and cytoplasm. Importantly, mutations in genes encoding nuclear envelope (NE) and associated proteins are the second highest cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in the heart remains poorly understood. METHODS AND RESULTS: We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or in adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue, and cellular analyses. High-resolution episcopic microscopy was used for three-dimensional reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided a physiological assessment of Lem2 iCKO adult mice. We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, reducing Lem2 levels to ∼45% in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age. CONCLUSIONS: Our data suggest that Lem2 is critical for integrity at the nascent NE in foetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, perhaps owing to a more established NE and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics.

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection.

Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

Messages from the voices within: regulation of signaling by proteins of the nuclear lamina.

The nuclear lamina (NL) is a protein scaffold lining the nuclear envelope that consists of nuclear lamins and associated transmembrane proteins. It helps to organize the nuclear envelope, chromosomes, and the cytoplasmic cytoskeleton. The NL also has an important role in regulation of signaling, as highlighted by the wide range of human diseases caused by mutations in the genes for NL proteins with associated signaling defects. This review will consider diverse mechanisms for signaling regulation by the NL that have been uncovered recently, including interaction with signaling effectors, modulation of actin assembly and compositional alteration of the NL. Cells with discrete NL mutations often show disruption of multiple signaling pathways, however, and for the most part the mechanistic basis for these complex phenotypes remains to be elucidated.

Luma is not essential for murine cardiac development and function.

AIMS: Luma is a recently discovered, evolutionarily conserved protein expressed in mammalian heart, which is associated with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex structurally integrates the nucleus and the cytoplasm and plays a critical role in mechanotransduction across the nuclear envelope. Mutations in several LINC components in both humans and mice result in various cardiomyopathies, implying they play essential, non-redundant roles. A single amino acid substitution of serine 358 to leucine (S358L) in Luma is the unequivocal cause of a distinct form of arrhythmogenic cardiomyopathy. However, the role of Luma in heart has remained obscure. In addition, it also remains to be determined how the S358L mutation in Luma leads to cardiomyopathy. METHODS AND RESULTS: To determine the role of Luma in the heart, we first determined the expression pattern of Luma in mouse heart. Luma was sporadically expressed in cardiomyocytes throughout the heart, but was highly and uniformly expressed in cardiac fibroblasts and vascular smooth muscle cells. We also generated germline null Luma mice and discovered that germline null mutants were viable and exhibited normal cardiac function. Luma null mice also responded normally to pressure overload induced by transverse aortic constriction. In addition, localization and expression of other LINC complex components in both cardiac myocytes and fibroblasts was unaffected by global loss of Luma. Furthermore, we also generated and characterized Luma S358L knock-in mice, which displayed normal cardiac function and morphology. CONCLUSION: Our data suggest that Luma is dispensable for murine cardiac development and function and that the Luma S358L mutation alone may not cause cardiomyopathy in mice.

A Survey of DDX21 Activity During Rev/RRE Complex Formation.

HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.

Nesprin 1α2 is essential for mouse postnatal viability and nuclear positioning in skeletal muscle.

The position of the nucleus in a cell is controlled by interactions between the linker of nucleoskeleton and cytoskeleton (LINC) complex and the cytoskeleton. Defects in nuclear positioning and abnormal aggregation of nuclei occur in many muscle diseases and correlate with muscle dysfunction. Nesprin 1, which includes multiple isoforms, is an integral component of the LINC complex, critical for nuclear positioning and anchorage in skeletal muscle, and is thought to provide an essential link between nuclei and actin. However, previous studies have yet to identify which isoform is responsible. To elucidate this, we generated a series of nesprin 1 mutant mice. We showed that the actin-binding domains of nesprin 1 were dispensable, whereas nesprin 1α2, which lacks actin-binding domains, was crucial for postnatal viability, nuclear positioning, and skeletal muscle function. Furthermore, we revealed that kinesin 1 was displaced in fibers of nesprin 1α2-knockout mice, suggesting that this interaction may play an important role in positioning of myonuclei and functional skeletal muscle.

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.

We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

Nuclear envelope protein Lem2 is required for mouse development and regulates MAP and AKT kinases.

The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease.

Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214.

UNLABELLED: In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV. IMPORTANCE: AdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

Targeted ablation of nesprin 1 and nesprin 2 from murine myocardium results in cardiomyopathy, altered nuclear morphology and inhibition of the biomechanical gene response.

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.

Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Host cell interactome of HIV-1 Rev includes RNA helicases involved in multiple facets of virus production.

The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Rev-interacting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. The strategy used here for identifying Rev interaction partners should prove effective for analyzing other viral and cellular proteins.

The structure of the NXF2/NXT1 heterodimeric complex reveals the combined specificity and versatility of the NTF2-like fold.

NXF1-like members of the NXF (nuclear export factor) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate-specific and tissue-specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NXT (NTF2-related export) protein is central to NXF family function; however, given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remain unclear. Here, we report the structural and functional analyses of the Caenorhabditis elegans NXF variant ceNXF2 bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity, including a probable site for phosphoregulation, have been identified. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for the colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.

Nuclear lamina at the crossroads of the cytoplasm and nucleus.

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.

DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.

Small molecule peptidomimetic inhibitors of importin α/ß mediated nuclear transport.

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the ß-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/ß pathway. By analyzing peptidomimetic libraries, we identified ß-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/ß but not by transportin. Structure-activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors.