Shuttling Receptor Research Articles

Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.

The Extraction Mechanism of Monoubiquitinated PEX5 from the Peroxisomal Membrane

The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6.

The peroxisomal matrix shuttling receptor Pex5 plays a role in FB1 production and virulence in Fusarium verticillioides

The peroxisomal matrix oxidase, catalase and peroxidase are imported peroxisomes through the shuttling receptors, which regulates the cellular oxidative homeostasis and function. Here, we report that PTS1 shuttling receptor FvPex5 is involved in the localization of PTS1, utilization of carbon sources and lipids, elimination ROS, cell wall stress, conidiation, fumonisin B1 (FB1) production, and virulence in maize pathogen Fusarium verticillioides. Significantly, differential expression of PTS1-, PTS2-, PEX- and FB1 toxin-related genes in wild type and ΔFvpex5 mutant were examined by RNA-Seq analyses and confirmed by RT-PCR assay. In addition, different expression of PTS1 and PTS2 genes of the ΔFvpex5 mutant were enriched in diverse biochemical pathways, such as carbon metabolism, nitrogen metabolism, lipid metabolism and the oxidation balance by combining GO and KEGG annotations. Overall, we showed that FvPex5 is involved in the regulation of genes associated with PTS, thereby affecting the oxidation balance, FB1 and virulence in F. verticillioides. The results help to clarify the functional divergence of Pex5 orthologs, and may provide a possible target for controlling F. verticillioides infections and FB1 biosynthesis.

Pex7 selectively imports PTS2 target proteins to peroxisomes and is required for anthracnose disease development in Colletotrichum scovillei

Pex7 is a shuttling receptor that imports matrix proteins with a type 2 peroxisomal targeting signal (PTS2) to peroxisomes. The Pex7-mediated PTS2 protein import contributes to crucial metabolic processes such as the fatty acid β-oxidation and glucose metabolism in a number of fungi, but cellular roles of Pex7 between the import of PTS2 target proteins and metabolic processes have not been fully understood. In this study, we investigated the functional roles of CsPex7, a homolog of the yeast Pex7, by targeted gene deletion in the pepper anthracnose fungus Colletotrichum scovillei. CsPex7 was required for carbon source utilization, scavenging of reactive oxygen species, conidial production, and disease development in C. scovillei. The expression of fluorescently tagged PTS2 signal of hexokinases and 3-ketoacyl-CoA thiolases showed that peroxisomal localization of the hexokinase CsGlk1 PTS2 is dependent on CsPex7, but those of the 3-ketoacyl-CoA thiolases are independent on CsPex7. In addition, GFP-tagged CsPex7 proteins were intensely localized to the peroxisomes on glucose-containing media, indicating a role of CsPex7 in glucose utilization. Collectively, these findings indicate that CsPex7 selectively recognizes specific PTS2 signal for import of PTS2-containing proteins to peroxisomes, thereby mediating peroxisomal targeting efficiency of PTS2-containing proteins in C. scovillei. On pepper fruits, the ΔCspex7 mutant exhibited significantly reduced virulence, in which excessive accumulation of hydrogen peroxide was observed in the pepper cells. We think the reduced virulence results from the abnormality in hydrogen peroxide metabolism of the ΔCspex7 mutant. Our findings provide insight into the cellular roles of CsPex7 in PTS2 protein import system.

Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin -Cout topology, and that PEX13 adopts a Nout -Cin topology, thus exposing its carboxy-terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. ENZYMES: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44.

Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii.

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

New insights into the distribution, protein abundance and subcellular localisation of the endogenous peroxisomal biogenesis proteins PEX3 and PEX19 in different organs and cell types of the adult mouse.

Peroxisomes are ubiquitous organelles mainly involved in ROS and lipid metabolism. Their abundance, protein composition and metabolic function vary depending on the cell type and adjust to different intracellular and environmental factors such as oxidative stress or nutrition. The biogenesis and proliferation of these important organelles are regulated by proteins belonging to the peroxin (PEX) family. PEX3, an integral peroxisomal membrane protein, and the cytosolic shuttling receptor PEX19 are thought to be responsible for the early steps of peroxisome biogenesis and assembly of their matrix protein import machinery. Recently, both peroxins were suggested to be also involved in the autophagy of peroxisomes (pexophagy). Despite the fact that distribution and intracellular abundance of these proteins might regulate the turnover of the peroxisomal compartment in a cell type-specific manner, a comprehensive analysis of the endogenous PEX3 and PEX19 distribution in different organs is still missing. In this study, we have therefore generated antibodies against endogenous mouse PEX3 and PEX19 and analysed their abundance and subcellular localisation in various mouse organs, tissues and cell types and compared it to the one of three commonly used peroxisomal markers (PEX14, ABCD3 and catalase). Our results revealed that the abundance of PEX3, PEX19, PEX14, ABCD3 and catalase strongly varies in the analysed organs and cell types, suggesting that peroxisome abundance, biogenesis and matrix protein import are independently regulated. We further found that in some organs, such as heart and skeletal muscle, the majority of the shuttling receptor PEX19 is bound to the peroxisomal membrane and that a strong variability exists in the cell type-specific ratio of cytosol- and peroxisome-associated PEX19. In conclusion, our results indicate that peroxisomes in various cell types are heterogeneous with regards to their matrix, membrane and biogenesis proteins.

Protein transport into peroxisomes: Knowns and unknowns.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.

The peroxisomal protein import machinery displays a preference for monomeric substrates.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

A potential role of karyopherin a2 in the impaired maturation of dendritic cells observed in glioblastoma patients

Aim: Patients with glioblastomas demonstrate well-documented immunological impairments including decreased numbers of mature dendritic cells (DCs). Recent data identified karyopherin a2 (KPNA2), a nucleocytoplasmic shuttling receptor, as diagnostic and prognostic biomarker for gliomas. The aim of this ongoing study is to correlate parameters of immunity and nucleocytoplasmic transport in glioblastoma patients. Methods: We preoperatively collected serum from 17 patients with glioblastomas and determined DC subsets (HLA DR+ Lin-, CD34-, CD45+, CD123+, CD11+ were analyzed) using a 6-color flow cytometry panel. Expression levels of KPNA2 and nuclear accumulation of p53 were evaluated semi-quantitatively by immunohistochemistry. O 6 -methylguanine DNA methyltransferase (MGMT) and isocitrate dehydrogenase-1 (IDH-1) status were assessed by pyrosequencing and immunohistochemistry, respectively. Results: Median expression levels for both KPNA2 and p53 were 5-10%. IDH-1-R132H mutation and MGMT promoter hypermethylation was detected in 3/16 and 1/9 patients, respectively. Mean counts of total mature DCs, myeloid DCs and plasmacytoid DCs were 9.6, 2.1, 3.4 cells/μL. A preliminary analysis suggests an association between low KPNA2 nuclear expression and increased numbers of mature DCs. However, this correlation did not reach statistical significance so far (P = 0.077). Conclusion: Our preliminary data may indicate a role of KPNA2 in the impaired maturation of DCs observed in glioblastoma patients.

Karyopherin a2 and chromosome region maintenance protein 1 expression in meningiomas: novel biomarkers for recurrence and malignant progression

The karyopherin protein family comprises importins and exportins which are nucleocytoplasmic shuttling receptors. Increased levels of karyopherin a2 and chromosome region maintenance protein 1 correlate with a higher WHO grade and a poorer prognosis in patients with infiltrative astrocytomas. The aim of this study was to evaluate representative members of importins and exportins (i.e. karyopherin a2 and chromosome region maintenance protein 1) as novel biomarkers for meningiomas of WHO grades I-III. We semiquantitatively analyzed nuclear expression of karyopherin a2, chromosome region maintenance protein 1 and the MIB1 labeling index using immunohistochemistry in 108 primary (44 meningiomas WHO grade I, 48 meningiomas WHO grade II, 16 meningiomas WHO grade III) and 13 recurrent meningiomas. Statistical analysis was performed using standard techniques. Karyopherin a2 (p < 0.001) and chromosome region maintenance protein 1 (p = 0.002) expression correlated significantly with the histological grade. Karyopherin a2 expression correlated with proliferative activity as assessed by the MIB1 index (p < 0.001). Recurrent tumors expressed significantly higher levels of karyopherin a2 (p = 0.045) when compared to primary growths. Multivariate analysis of the overall series as well as of patients with atypical meningiomas identified higher karyopherin a2 (≥ 5 vs. <5%) and chromosome region maintenance protein 1 (≥ 60 vs. 60%) expression as independent predictors of tumor recurrence. Karyopherin a2 and chromosome region maintenance protein 1 expression may have potential as novel biomarkers for meningiomas.

A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Ubiquitin in the peroxisomal protein import pathway

PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation.

Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys‐Monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.

Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1

BackgroundThe proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.ResultsTo shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.ConclusionsThese studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.

The Dynamin-like Protein Vps1p of the Yeast Saccharomyces cerevisiae Associates with Peroxisomes in a Pex19p-dependent Manner

Dynamins and dynamin-like proteins play important roles in organelle division. In Saccharomyces cerevisiae, the dynamin-like protein Vps1p (vacuolar protein sorting protein 1) is involved in peroxisome fission, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes. What relationship Vps1p has with peroxisomes remains unclear. Here we show that Vps1p interacts with Pex19p, a peroxin that acts as a shuttling receptor for peroxisomal membrane proteins or as a chaperone assisting the assembly/stabilization of proteins at the peroxisome membrane. Vps1p contains two putative Pex19p recognition sequences at amino acids 509-523 and 633-647. Deletion of the first (but not the second) sequence results in reduced numbers of enlarged peroxisomes in cells, as in vps1delta cells. Deletion of either sequence has no effect on vacuolar morphology or vacuolar protein sorting, suggesting that the peroxisome and vacuole biogenic functions of Vps1p are separate and separable. Substitution of proline for valine at position 516 of Vps1p abrogates Pex19p binding and gives the peroxisome phenotype of vps1delta cells. Microscopic analysis showed that overexpression of Pex19p or redirection of Pex19p to the nucleus does not affect the normal cellular distribution of Vps1p in the cytosol and in punctate structures that are not peroxisomes, suggesting that Pex19p does not function in targeting Vps1p to peroxisomes. Subcellular fractionation showed that a fraction of Vps1p is associated with peroxisomes and that deletion or mutation of the first Pex19p recognition sequence abrogates this association. Our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to peroxisomes.

Deciphering networks of protein interactions at the nuclear pore complex.

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

Regulated nuclear import of the STAT1 transcription factor by direct binding of importin-alpha.

Signal transducers and activators of transcription (STATs) reside in a latent state in the cytoplasm of the cell, but accumulate in the nucleus in response to cytokines or growth factors. Localization in the nucleus occurs following STAT tyrosine phosphorylation and dimerization. In this report we demonstrate a direct interaction of importin-alpha5 with tyrosine-phosphorylated STAT1 dimers, and provide evidence that a nuclear localization signal (NLS) exists in an inactive state within a STAT1 monomer. A mutation in STAT1 leucine 407 (L407A) is characterized, which generates a protein that is accurately tyrosine phosphorylated in response to interferon, dimerizes and binds DNA, but does not localize to the nucleus. The import defect of STAT1(L407A) appears to be a consequence of the inability of this protein to be recognized by its import shuttling receptor. In addition, we demonstrate that STAT1 binding to specific target DNA effectively blocks importin-alpha5 binding. This result may play a role in localizing STAT1 to its destination in the nucleus, and in releasing importin-alpha5 from STAT1 for recycling back to the cytoplasm.

Analysis of nuclear protein import and export in vitro using fluorescent cargoes.

AbstractNucleocytoplasmic transport of protein is central to the regulation of many cellular functions. Nuclear import and export takes place through nuclear pore complexes (NPCs; reviewed in refs. 1 and 2), large supramolecular structures that span the nuclear envelope. Cargo proteins contain specific signals for nuclear transport, which are often short stretches of amino acids. Nuclear localization signals (NLSs) direct the nuclear import of proteins, whereas nuclear export sequences (NESs) specify protein export from the nucleus. Both types of signals are recognized by nucleocytoplasmic shuttling receptor proteins belonging to the importin/karyopherin β family (reviewed in refs. 3 and 4). After binding to cargo, these receptors interact with proteins of the NPC to mediate cargo translocation into or out of the nucleus. The best-characterized nuclear-import pathway utilizes the major import-receptor importin β, which mediates the transport of proteins with basic amino-acid-rich NLSs. Importin (3 interacts with cargo molecules either directly (5) or via an adapter protein, importin α (3). Export of many proteins from the nucleus is mediated by the export receptor chromosome region maintenance 1 (CRM1), which recognizes leucine-rich NESs (6-10).KeywordsPermeabilized CellNuclear ExportNuclear ImportTransport ReactionNuclear TransportThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.