Comparison of diverse transport signals in synthetic peptide-induced nuclear transport

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.     

A protein recognized by antibodies to Asp-Asp-Asp-Glu-Asp shows specific binding activity to heterogeneous nuclear transport signals

Nuclear proteins contain a signal, termed the nuclear transport signal, that specifies their selective transport into the nucleus. Previously we reported that antibodies to Asp-Asp-Asp-Glu-Asp (DDDED) inhibited nuclear transport of nuclear proteins in vivo. We therefore tried to detect a cellular receptor of nuclear transport signals as a protein that reacted with both anti-DDDED antibody and nuclear transport signal sequences. Using two steps of affinity chromatography, anti-DDDED-Sepharose and nucleoplasmin-Sepharose, we obtained a protein of 69 kDa (p69) from the nuclear pore fraction that showed these characters. This p69 recognized by anti-DDDED antibody interacted specifically with SV40 large T antigen and nucleoplasmin transport signals.     

The nucleocytoplasmic transport of viral proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.     

Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.     

An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport

Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery.     

Dynamic localization of the nuclear import receptor and its interactions with transport factors

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin- beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal- bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.     

Nucleocytoplasmic shuttling signals: two for the price of one

It has been appreciated for some time that basic-amino-acid-type nuclear localization signals control nuclear uptake of proteins and that leucine-rich nuclear export signals mediate export back into the cytoplasm. The machinery that recognizes and escorts these well-defined protein transport signals through the nuclear pore complex has been identified and characterized. Does this mean that the nuclear transport field knows all it needs to about transport signals? Not quite, as several recent publications have expanded the membership of a growing family of transport signals, known as nucleocytoplasmic shuttling (NS) signals. All proteins currently known to contain this type of signal also associate with mRNA. This article reviews what is currently known about mediators of NS signal transport and discusses the link between NS signal-containing proteins and RNA export.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.     

Qualitative highly divergent nuclear export signals can regulate export by the competition for transport cofactors in vivo

Nucleo-cytoplasmic transport of proteins is mediated by nuclear export signals, identified in various proteins executing heterologous biological functions. However, the molecular mechanism underlying the orchestration of export is only poorly understood. Using microinjection of defined recombinant export substrates, we now demonstrate that leucine-rich nuclear export signals varied dramatically in determining the kinetics of export in vivo . Thus, nuclear export signals could be kinetically classified which correlated with their affinities for CRM1-containing export complexes in vitro . Strikingly, cotransfection experiments revealed that proteins containing a fast nuclear export signal inhibited export and the biological activity of proteins harboring a slower nuclear export signal in vivo . The affinity for export complexes seems therefore predominantly controlled by the nuclear export signal itself, even in the context of the complete protein in vivo . Overexpression of FG-rich repeats of nucleoporins affected a medium nuclear export signal containing protein to the same extent as a fast nuclear export signal containing protein, indicating that nucleoporins appear not to contribute significantly to nuclear export signal-specific export regulation. Our results imply a novel mode for controlling the biological activity of shuttle proteins already by the composition of the nuclear export signal itself.     

Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe

The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described.     

The effects of variations in the number and sequence of targeting signals on nuclear uptake

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.     

Cse1p-binding dynamics reveal a binding pattern for FG-repeat nucleoporins on transport receptors

Nuclear pore proteins with phenylalanine-glycine repeats are vital to the functional transport of molecules across the nuclear pore complex. The current study investigates the binding of these FG-nucleoporins to the Cse1p:Kap60p:RanGTP nuclear export complex. Fourteen binding spots for FG-nucleoporin peptides are revealed on the surface of Cse1p, and 5 are revealed on the Kap60p surface. Taken together, and along with binding data for two other transport receptors, the data suggest that the ability to bind FG-nucleoporins by itself is not enough to ensure viable nuclear transport. Rather, it is proposed that the density of binding spots on the transport receptor surface is key in determining transport viability. The number of binding spots on the transport receptor surface should be large enough to ensure multiple, simultaneous FG-repeat binding, and their arrangement should be close enough to ensure multiple binding from the same FG-nucleoporin.     

Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.     

Active transport of proteins into the nucleus

Nuclear proteins are actively and posttranslationally transported across the nuclear envelope. This transport is a highly selective process that can be divided into two steps, receptor-binding followed by translocation through the nuclear envelope. Receptor-binding is mediated by nuclear localization signals that have been identified in many nuclear proteins. Translocation is energy-dependent and occurs through the nuclear pore complex.     

A multimeric membrane protein reveals 14-3-3 isoform specificity in forward transport in yeast

Arginine (Arg)-based endoplasmic reticulum (ER) localization signals are sorting motifs involved in the quality control of multimeric membrane proteins. They are distinct from other ER localization signals like the C-terminal di-lysine [-K(X)KXX] signal. The Pmp2p isoproteolipid, a type I yeast membrane protein, reports faithfully on the activity of sorting signals when fused to a tail containing either an Arg-based motif or a -KKXX signal. This reporter reveals that the Arg-based ER localization signals from mammalian Kir6.2 and GB1 proteins are functional in yeast. Thus, the machinery involved in recognition of Arg-based signals is evolutionarily conserved. Multimeric presentation of the Arg-based signal from Kir6.2 on Pmp2p results in forward transport, which requires 14-3-3 proteins encoded in yeast by BMH1 and BMH2 in two isoforms. Comparison of a strain without any 14-3-3 proteins (Deltabmh2) and the individual Deltabmh1 or Deltabmh2 shows that the role of 14-3-3 in the trafficking of this multimeric Pmp2p reporter is isoform-specific. Efficient forward transport requires the presence of Bmh1p. The specific role of Bmh1p is not due to differences in abundance or affinity between the isoforms. Our results imply that 14-3-3 proteins mediate forward transport by a mechanism distinct from simple masking of the Arg-based signal.     

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

Identification and characterization of nuclear location signal-binding proteins in nuclear envelopes

A radioiodinated, photoactivable synthetic nonapeptide corresponding to the nuclear location signal (NLS) of SV40 large T antigen has been used in photolabelling reactions with purified mouse liver nuclei, nuclear envelopes and other cellular fractions, to identify specific NLS-binding proteins which may be involved in selective transport of karyophilic proteins. SDS-polyacrylamide gel analysis of photolabelled products demonstrates that a 60 kDa nuclear protein and four nuclear envelope proteins (67, 60, 53 and 47 kDa) bind specifically to the native NLS and not to a mutant NLS or unrelated sequences. This binding shows saturation kinetics, with highest affinity of the NLS for the 60 and 67 kDa proteins. The nuclear 60 kDa NLS-binding protein is identical to the nuclear envelope 60 kDa NLS-binding protein by two-dimensional gel analysis of labelled proteins. Biochemical fractionation of labelled nuclear envelopes suggests that the 53 and 47 kDa proteins are peripheral membrane proteins whereas the 67 and 60 kDa proteins can be localized to the pore complex. The NLS also binds to solubilized 67, 60, 53 and 47 kDa proteins but with decreased affinity. Our results suggest that one of the early steps in selective nuclear transport of proteins may be the recognition of the NLS by the 60 kDa and/or 67 kDa binding proteins present in the nuclear pore complex.     

Functional Tat transport of unstructured, small, hydrophilic proteins

The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.