scholarly journals Reengineering Ribosome Export

2009 ◽  
Vol 20 (5) ◽  
pp. 1545-1554 ◽  
Author(s):  
Kai-Yin Lo ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Large Subunit ◽  
Nuclear Export Signal ◽  
Ribosomal Subunit ◽  
Nuclear Pore ◽  
Multiple Receptors ◽  
Ribosomal Subunit Protein ◽  
Specific Receptors ◽  
Ribosome Export ◽  
Export Receptors

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.

scholarly journals Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

2010 ◽  
Vol 189 (7) ◽  
pp. 1079-1086 ◽  
Author(s):  
Jayati Sengupta ◽  
Cyril Bussiere ◽  
Jesper Pallesen ◽  
Matthew West ◽  
Arlen W. Johnson ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Structural Data ◽  
Release Mechanism ◽  
Nuclear Pore ◽  
Subunit Joining

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

scholarly journals The SUMO-specific isopeptidase SENP2 associates dynamically with nuclear pore complexes through interactions with karyopherins and the Nup107-160 nucleoporin subcomplex

2011 ◽  
Vol 22 (24) ◽  
pp. 4868-4882 ◽  
Author(s):  
Jacqueline Goeres ◽  
Pak-Kei Chan ◽  
Debaditya Mukhopadhyay ◽  
Hong Zhang ◽  
Brian Raught ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Localization Signal ◽  
Import And Export ◽  
Pore Complexes ◽  
Eukaryotic Organisms ◽  
Export Receptors

The association of small, ubiquitin-related modifier–specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat–containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

scholarly journals The Nuclear Export Receptors TbMex67 and TbMtr2 Are Required for Ribosome Biogenesis in Trypanosoma brucei

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Constance Rink ◽  
Martin Ciganda ◽  
Noreen Williams
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Biological Process ◽  
Large Subunit ◽  
Critical Role ◽  
Ribosomal Subunits ◽  
Content Type ◽  
Protein Components ◽  
Export Receptors

ABSTRACT Ribosomal maturation is a complex and highly conserved biological process involving migration of a continuously changing RNP across multiple cellular compartments. A critical point in this process is the translocation of individual ribosomal subunits (60S and 40S) from the nucleus to the cytoplasm, and a number of export factors participate in this process. In this study, we characterize the functional role of the auxiliary export receptors TbMex67 and TbMtr2 in ribosome biogenesis in the parasite Trypanosoma brucei. We demonstrate that depletion of each of these proteins dramatically impacts the steady-state levels of other proteins involved in ribosome biogenesis, including the trypanosome-specific factors P34 and P37. In addition, we observe that the loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation. Although the TbMex67-TbMtr2 heterodimer is structurally distinct from Mex67-Mtr2 complexes previously studied, our data show that they retain a conserved function in ribosome biogenesis. IMPORTANCE The nuclear export of ribosomal subunits (60S and 40S) depends in part on the activity of the essential auxiliary export receptors TbMtr2 and TbMex67. When these proteins are individually depleted from the medically and agriculturally significant parasite Trypanosoma brucei, distinct alterations in the processing of the rRNAs of the large subunit (60S) are observed as well as aberrations in the assembly of functional ribosomes (polysomes). We also established that TbMex67 and TbMtr2 interact directly or indirectly with the protein components of the 5S RNP, including the trypanosome-specific P34/P37. The critical role that TbMex67 and TbMtr2 play in this essential biological process together with their parasite-specific interactions may provide new therapeutic targets against this important parasite.

scholarly journals Mutational Analysis of Bovine Leukemia Virus Rex: Identification of a Dominant-Negative Inhibitor

2005 ◽  
Vol 79 (11) ◽  
pp. 7172-7181 ◽  
Author(s):  
Eun-A Choi ◽  
Thomas J. Hope
Keyword(s):  
Nuclear Localization ◽  
Bovine Leukemia Virus ◽  
Nuclear Export ◽  
Protein Localization ◽  
Leukemia Virus ◽  
Nuclear Export Signal ◽  
Dominant Negative ◽  
Nuclear Pore ◽  
Large Animal ◽  
Wild Type

ABSTRACT The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model.

scholarly journals Observation of Single Nuclear Export Receptors at the Nuclear Pore Complex

2010 ◽  
Vol 98 (3) ◽  
pp. 307a
Author(s):  
Ulrike Schmitz-Ziffels ◽  
Andreas Veenendaal ◽  
Jan Peter Siebrasse ◽  
Ulrich Kubitscheck
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Export Receptors

scholarly journals Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors

2016 ◽  
Vol 36 (24) ◽  
pp. 3019-3032 ◽  
Author(s):  
Anne-Marie Landry-Voyer ◽  
Sarah Bilodeau ◽  
Danny Bergeron ◽  
Kiersten L. Dionne ◽  
Sarah A. Port ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Nuclear Export Signal ◽  
Ribosomal Subunit ◽  
Stable Complex ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Late Maturation

Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3′-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L -null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.

scholarly journals Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress

2021 ◽  
Author(s):  
Ukrae H. Cho ◽  
Martin W. Hetzer
Keyword(s):  
Endoplasmic Reticulum ◽  
Focal Adhesion ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Genome Regulation ◽  
Focal Adhesion Proteins ◽  
Domain Protein ◽  
Export Signal

Introductory ParagraphDuring programmed cell death, caspases degrade 7 out of ∼30 nucleoporins (Nups) to irreversibly demolish the nuclear pore complex (NPC)1. However, for poorly understood reasons, caspases are also activated in differentiating cells in a non-apoptotic manner2,3. Here, we describe reversible, caspase-mediated NPC “trimming” during early myogenesis. We find that sublethal levels of caspases selectively proteolyze 4 peripheral Nups, Nup358, Nup214, Nup153, and Tpr, resulting in the transient block of nuclear export pathways. Several nuclear export signal (NES)-containing focal adhesion proteins concomitantly accumulate in the nucleus where they function as transcription cofactors4. We show that one such protein, FAK (focal adhesion kinase), drives a global reconfiguration of MBD2 (methyl CpG binding domain protein 2)-mediated genome regulation. We also observe caspase-mediated NPC trimming during neurogenesis and endoplasmic reticulum (ER) stress. Our results illustrate that the NPC can be proteolytically regulated in response to non-apoptotic cues, and call for a reassessment of the death-centric view of caspases.

scholarly journals Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

2008 ◽  
Vol 19 (2) ◽  
pp. 735-744 ◽  
Author(s):  
Nai-Jung Hung ◽  
Kai-Yin Lo ◽  
Samir S. Patel ◽  
Kara Helmke ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Yeast Two Hybrid ◽  
Synthetic Lethal ◽  
Nuclear Export Receptor ◽  
Export Receptors ◽  
Two Hybrid

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

scholarly journals The nuclear export factor Xpo1p targets Mad1p to kinetochores in yeast

2009 ◽  
Vol 184 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Robert J. Scott ◽  
Lucas V. Cairo ◽  
David W. Van de Vosse ◽  
Richard W. Wozniak
Keyword(s):  
Signaling Pathway ◽  
Nuclear Export ◽  
Spindle Assembly Checkpoint ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Guanosine Triphosphate ◽  
Intranuclear Transport ◽  
Pore Complexes ◽  
Export Signal

Nuclear pore complexes (NPCs) mediate all nucleocytoplasmic traffic and provide docking sites for the spindle assembly checkpoint (SAC) protein Mad1p. Upon SAC activation, Mad1p is recruited onto kinetochores and rapidly cycles between NPCs and kinetochores. We examined the mechanism of Mad1p movement onto kinetochores and show that it is controlled by two components of the nuclear transport machinery, the exportin Xpo1p and Ran–guanosine triphosphate (GTP). Mad1p contains a nuclear export signal (NES) that is recognized by Xpo1p. The NES, Xpo1p, and RanGTP are all required for Mad1p recruitment onto kinetochores in checkpoint-activated cells. Consistent with this function, Xpo1p also accumulates on kinetochores after SAC activation. We have also shown that Xpo1p and RanGTP are required for the dynamic cycling of Mad1p between NPCs and kinetochores in checkpoint-arrested cells. These results reveal an important function for Xpo1p in mediating intranuclear transport events and identify a signaling pathway between kinetochores and NPCs.

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