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Implications for Proteasome Nuclear Localization Revealed by the Structure of the Nuclear Proteasome Tether Protein Cut8

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Implications for Proteasome Nuclear Localization Revealed by the Structure of the Nuclear Proteasome Tether Protein Cut8 Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8 Kojiro Takedaa, Nam K. Tonthatb, Tiffany Gloverc, Weijun Xuc, Eugene V. Koonind, Mitsuhiro Yanagidaa, and Maria A. Schumacherb,1 aG0 Cell Unit; Okinawa Institute of Science and Technology (OIST); 1919-1 Tancha, Onna, Okinawa, Japan; bDepartment of Biochemistry, Duke University Medical Center, Room 243A Nanaline H. Duke Building, Box 3711, Durham, NC 27710; cDepartment of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Unit 1000, Houston, TX 77030; and dNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20892 Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved August 29, 2011 (received for review March 7, 2011) Degradation of nuclear proteins by the 26S proteasome is essential ultimately found to be a delay in the destruction of the mitotic for cell viability. In yeast, the nuclear envelope protein Cut8 med- cyclin and securin (13). However, the polyubiquitination of these iates nuclear proteasomal sequestration by an uncharacterized proteins was normal, suggesting a role for the proteasome. Sub- mechanism. Here we describe structures of Schizosaccharomyces sequent studies showed that Cut8 is responsible for sequestering pombe Cut8, which shows that it contains a unique, modular the 26S proteasome to the nucleus (10, 11). Cut8 was found to be fold composed of an extended N-terminal, lysine-rich segment localized to the nuclear envelope and overlapping the location that when ubiquitinated binds the proteasome, a dimer domain of the proteasome (17–21). An interaction between Cut8 and followed by a six-helix bundle connected to a flexible C tail. The the proteasome was subsequently demonstrated (11). Additional Cut8 six-helix bundle shows structural similarity to 14-3-3 phos- data showing that Cut8 is essential for nuclear localization is the phoprotein-binding domains, and binding assays show that this recent finding that a reduction in Cut8 levels coincides with the domain is necessary and sufficient for liposome and cholesterol altered localization of the proteasome from the nucleus to the binding. Moreover, specific mutations in the 14-3-3 regions corre- cytoplasm upon the transition from vegetative proliferation to sponding to putative cholesterol recognition/interaction amino the G0/quiescent phase (22). Nuclear sequestration of the protea- acid consensus motifs abrogate cholesterol binding. In vivo studies some by Cut8 has also been shown to be critical for double-strand confirmed that the 14-3-3 region is necessary for Cut8 membrane break repair (23). This is likely due to the requirement of the localization and that dimerization is critical for its function. Thus, proteasome for cohesion cleavage. Thus, not only is Cut8 crucial the data reveal the Cut8 organization at the nuclear envelope. for normal anaphase progression because it ensures essential Reconstruction of Cut8 evolution suggests that it was present in anaphase-promoting proteolytic events in the nucleus, but it is the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic also required for overall genome stability due to its function in feature. The importance of Cut8 for cell viability and its absence DNA repair events. in humans suggests it as a possible target for the development Interestingly, Cut8 tethers the proteasome in the nucleus of specific chemotherapeutics against invasive fungal infections. through ubiquitinated lysine residues within its N-terminal re- gion. In addition to conferring tight binding of Cut8 to the 26S proteasome, ubiquitination also results in a short half-life of the he 26S proteasome is a large supramolecular machine that Tcarries out essential ubiquitin-mediated proteolysis events in Cut8 protein (approximately 3 min) as Cut8 itself is ultimately all eukaryotes (1–3). The importance of the proteasomal degra- a substrate of the proteasome. Takeda and Yanagida proposed dation of cytosolic proteins, such as antigens for presentation by that the short-lived nature of Cut8 is critical for feedback enrich- major histocompatibility complex molecules, is well known (4). ment of proteasome inside the nucleus, allowing Cut8 to act as a However, a large number of nuclear proteins also acquire the proteasome sensor (11). Given its central role in essentially all proteasomal-targeting polyubiquination tag as a consequence of cellular processes, the way(s) in which the proteasome is localized ubiquitin-activating, ubiquitin-conjugating, and ubiquitin-ligating within cells is of great importance. Cut8 represents the best char- enzymes (5). Nuclear proteins that are degraded in this manner acterized proteasome anchor protein. However, the molecular by the proteasome include the securin protein, which cleaves co- mechanism by which Cut8 binds and retains the proteasome is hesin to allow proper chromosome segregation (6–8). Although unknown, and Cut8 shows no homology to any protein of known ubiquitin-mediated proteasomal degradation of nuclear proteins structure. To gain insight into Cut8 function, we carried out struc- plays key roles in cell viability, the mechanisms by which the pro- tural, biochemical, and in vivo functional studies on the S. pombe teasome is sequestered in the nucleus is still not well understood. Cut8 protein. In the fungi Schizosaccharomyces pombe, data clearly demon- strated that the proteasome is enriched in the nucleus and, prin- Author contributions: K.T. and M.A.S. designed research; K.T., N.K.T., T.G., W.X., and M.A.S. cipally, the nuclear envelope (9). This localization was found to performed research; K.T., E.V.K., M.Y., and M.A.S. analyzed data; and M.A.S. wrote be mediated by the nuclear envelope protein, Cut8 (10, 11). the paper. Cut8 was originally identified in fission yeast as a tempera- The authors declare no conflict of interest. – ture-sensitive mutant, Cut8 563, that gives rise to the cut or cell This article is a PNAS Direct Submission. untimely torn phenotype (12, 13). cut8 mutants do not complete Freely available online through the PNAS open access option. mitosis and have hypercondensed chromosomes and a short Data deposition: The atomic coordinates for the P1 and C2 Cut8 structures have been spindle (13). This phenotype is similar to that displayed by yeast deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3Q5W and 3Q5X, that are defective in 26S proteasome components (14–16). In respectively). both cut8 and proteasome mutants, chromosome missegregation 1To whom correspondence should be addressed. E-mail: [email protected]. and aberrant spindle dynamics occur, but cytokinesis is normal, This article contains supporting information online at www.pnas.org/lookup/suppl/ leading to the cut phenotype. The cause of this phenotype was doi:10.1073/pnas.1103617108/-/DCSupplemental. 16950–16955 ∣ PNAS ∣ October 11, 2011 ∣ vol. 108 ∣ no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1103617108 Downloaded by guest on September 26, 2021 Results and Discussion ubiquitination of four lysine residues in the N-terminal segment: Structure Determination of S. pombe Cut8. For structural and bio- Lys10, Lys11, Lys13, and Lys22. Whereas Lys10, Lys11, and Lys13 chemical studies an artificial S. pombe cut8 gene, codon-opti- form part of the disordered region in the Cut8 structure, Lys22 mized for expression in Escherichia coli, was utilized (SI Materials is visible and resides in the solvent exposed loop region of the and Methods). The full-length (FL) Cut8 protein was susceptible N-terminal arm (Fig. 1C). Interestingly, the ubiquitination of to proteolysis, and mass spectroscopic analyses revealed that it these lysines also serves as a degron tag for eventual destruction degraded to a stable fragment corresponding to residues 1–217. by the proteasome. Hence, the extended, flexible nature of the Therefore, Cut8(1–217) was produced for structural studies and Cut8 N-terminal region is consistent with its role as a proteasome two crystal forms, P1 and C2, were obtained. The P1 crystal form binding and sensor module. – was solved by multiple wavelength anomalous diffraction (MAD) The second domain of Cut8, residues 32 71, is composed of α (Fig. 1A). The structure contains two Cut8 molecules in the crys- three short -helices that form an intimate dimer connecting – tallographic asymmetric unit (ASU) and the final model includes regions 1 and 3. Domain 3, from residues 72 216, forms a large, residues 18–216 of one subunit, 19–216 of the second subunit, antiparallel six-helix bundle, which extends more than 40 Å below R ∕R the dimer domain. Cut8 is a domain-swapped dimer, whereby and has an work free of 23.1%/27.6% to 2.75-Å resolution (24–26). The C2 crystal form was solved by Molecular Replace- domain 3 of one subunit interacts with the N-terminal arm of ment using the P1 structure as a search model and contains the other subunit to specifically affix and orient the arm (Fig. 1C). one Cut8 subunit in the ASU (Fig. 1B). The C2 model includes Our proteolysis studies indicate that the C-terminal region, from – R ∕R residues 217–262, is disordered or highly flexible. Cut8 residues 25 216 and has been refined to an work free of 26.8%/29.4% to 2.98-Å resolution (Table S1). Cut8 Forms an Intertwined Dimer. Although previous studies using GST pull downs showed that Cut8 interacts with itself, the specific Cut8 Contains a Unique, Modular Fold. The structures show that Cut8 is an all-helical protein with the topology (α1; residues oligomeric state of Cut8 has been unknown (11). The crystal struc- ture revealed the presence of an extensive dimer, which buries 35–42, α2; 46–57, α3; 60–69, α4; 75–92, α5; 105–125, 310; 126– 2 4;995 Å of protein surface from solvent (Fig. 1D). This is a unique 129, α6; 135–149, α7; 156–181, α8; 190–201, α9; 203–214).
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