Dataset Information

Nuclear export of ubiquitinated proteins via the UBIN-POST system.

ABSTRACT: Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

SUBMITTER: Hirayama S

PROVIDER: S-EPMC5939056 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Nuclear export of ubiquitinated proteins via the UBIN-POST system.

Hirayama Shoshiro S Sugihara Munechika M Morito Daisuke D Iemura Shun-Ichiro SI Natsume Tohru T Murata Shigeo S Nagata Kazuhiro K

Proceedings of the National Academy of Sciences of the United States of America 20180416 18

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinat ...[more]

PMID: 29666234

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Project description:Fluorescent protein biosensors are powerful cellular systems biology tools for dissecting the complexity of cellular processes with high spatial and temporal resolution. As regulated nucleo-cytoplasmic transport is crucial for the modulation of numerous (patho)physiological cellular responses, a detailed understanding of its molecular mechanism would open up novel options for a rational manipulation of the cell. In contrast to genetic approaches, we here established and employed high-content cellular translocation biosensors applicable for dissecting nuclear export by chemicogenomics. A431 cell lines, stably expressing a translocation biosensor composed of glutathione S-transferase, GFP and a rational combination of nuclear import and export signals, were engineered by antibiotic selection and flow cytometry sorting. Using an optimized nuclear translocation algorithm, the translocation response could be robustly quantified on the Cellomics Arrayscan(®) VTI platform. Subsequent to assay optimization, the assay was developed into a higher density 384-well format high-content assay and employed for the screening of the 17K ChemBioNet compound collection. This library was selected on the basis of a genetic algorithm used to identify maximum common chemical substructures in a database of annotated bioactive molecules and hence, is well-placed in the chemical space covered by bioactive compounds. Automated multiparameter data analysis combined with visual inspection allowed us to identify and to rationally discriminate true export inhibitors from false positives, which included fluorescent compounds or cytotoxic substances that dramatically affected the cellular morphology. A total of 120 potential hit compounds were selected for Cellomics Arrayscan(®) VTI based rescreening. The export inhibitory activity of 20 compounds effective at concentrations < 25 μM were confirmed by fluorescence microscopy in several cell lines. Interestingly, kinetic analysis allowed the identification of inhibitors capable to interfere with the export receptor CRM1-mediated nuclear export not only in an irreversible, but also in a reversible fashion. In sum, exploitation of biosensor based screening allows the identification of chemicogenomic tools applicable for dissecting nucleo-cytoplasmic transport in living cells.

Dataset Information

Nuclear export of ubiquitinated proteins via the UBIN-POST system.

Publications

Nuclear export of ubiquitinated proteins via the UBIN-POST system.

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