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. 2015 Sep 11;290(37):22678-85.
doi: 10.1074/jbc.M115.673038. Epub 2015 Jul 28.

Pli1(PIAS1) SUMO ligase protected by the nuclear pore-associated SUMO protease Ulp1SENP1/2

Minghua Nie  1 , Michael N Boddy  2
Affiliations

Pli1(PIAS1) SUMO ligase protected by the nuclear pore-associated SUMO protease Ulp1SENP1/2

Minghua Nie et al. J Biol Chem. .

Abstract

Covalent modification of the proteome by SUMO is critical for genetic stability and cell growth. Equally crucial to these processes is the removal of SUMO from its targets by the Ulp1 (HuSENP1/2) family of SUMO proteases. Ulp1 activity is normally spatially restricted, because it is localized to the nuclear periphery via interactions with the nuclear pore. Delocalization of Ulp1 causes DNA damage and cell cycle defects, phenotypes thought to be caused by inappropriate desumoylation of nucleoplasmic targets that are normally spatially protected from Ulp1. Here, we define a novel consequence of Ulp1 deregulation, with a major impact on SUMO pathway function. In fission yeast lacking Nup132 (Sc/HuNUP133), Ulp1 is delocalized and can no longer antagonize sumoylation of the PIAS family SUMO E3 ligase, Pli1. Consequently, SUMO chain-modified Pli1 is targeted for proteasomal degradation by the concerted action of a SUMO-targeted ubiquitin ligase (STUbL) and Cdc48-Ufd1-Npl4. Pli1 degradation causes the profound SUMO pathway defects and associated centromere dysfunction in cells lacking Nup132. Thus, perhaps counterintuitively, Ulp1-mediated desumoylation can promote SUMO modification by stabilizing a SUMO E3 ligase.

Keywords: Cdc48-Ufd1-Npl4; Pli1; STUbL; Ulp1; nuclear pore; proteasome; protein degradation; small ubiquitin-like modifier (SUMO); sumoylation.

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Figures

FIGURE 1.
FIGURE 1.
Nup132 deletion suppresses lethality and accumulation of SUMO conjugates in slx8-29 and ulp2Δ mutants. A, serial dilutions of the indicated strains were spotted onto media with indicated drugs, grown at the indicated temperatures. B, Western blots of sumoylated proteins or tubulin of indicated strains grown at 25 °C to log phase or to mid log phase then shifted to 35 °C for 6 h. The black arrowhead indicates the position of free mature SUMO.
FIGURE 2.
FIGURE 2.
Cells lacking Nup132 have altered Ulp1 activity. A, live cell imaging of GFP fluorescence of Ulp1-GFP and DAPI staining in wild type (top panels) and nup132Δ cells (bottom panels). In addition to defining the nucleus, live cell staining of fission yeast with DAPI also delineates the cell wall and septum. B, Western blots of Ulp1 endogenously tagged with Myc13 or tubulin of indicated strains. The black arrowhead points to the position of full-length Ulp1-Myc13. The asterisk marks the position of a truncated form of Ulp1-Myc13. C, Western blots of sumoylated proteins or tubulin of indicated strains. Ectopic expression of SUMOGG or SUMOFL under the nmt41 promoter was induced by growing cells in the absence of thiamine (B1) for 22 h at 25 °C and then an additional 6 h at 35 °C. The black arrowhead indicates the position of free mature SUMO, and the white arrowhead indicates the position of immature SUMO.
FIGURE 3.
FIGURE 3.
Pli1 SUMO ligase is destabilized in nup132Δ cells. A, wild type or nup132Δ cells grown at 25 °C to mid log phase and then shifted to 35 °C for 4 h. Cycloheximide was added to cell cultures at 200 μg/ml, and cells were harvested at 0, 15, 30, 60, or 120 min after cycloheximide addition and analyzed by Western blotting. The levels of Pli1-TAP proteins (unmodified and all SUMO modified species) were normalized to the levels of tubulin in each sample and quantified with National Institutes of Health ImageJ software. The graph is representative of results from three experiments. B, Western blots of Pli1-TAP or tubulin of indicated strains grown at 25 °C to log phase (top panels) or to mid log phase then shifted to 35 °C for 6 h (bottom panels). Black and white arrowheads mark the positions of unmodified and SUMO modified Pli1-TAP bands, respectively.
FIGURE 4.
FIGURE 4.
Pli1 SUMO ligase is stabilized by Ulp1-mediated desumoylation. A, His6-SUMO was expressed from the nmt41 promoter in slx8-29 nup132Δ cells. Cells were grown in the absence of B1 for 24 h, and proteins were extracted by TCA and pulled down on nickel-nitrilotriacetic acid (Ni) beads. Input (IN), pulldown (PD), and flow through (FT) were analyzed for the presence of Pli1-TAP by peroxidase anti-peroxidase (PAP) Western blotting. B, Western blots of Pli1-TAP or tubulin of indicated strains grown at 25 °C to log phase. C, cells were grown in the absence of B1 for 24 h to induce the ectopic expression of nmt41-driven Ulp1. In the A–C, black and white arrowheads mark the position of unmodified and modified Pli1-TAP bands. D, the transcript levels of ulp1 and pli1 of cells grown in the absence of B1 for 24 h to induce the ectopic expression of nmt41-driven Ulp1 or under repressed condition (+ B1) were analyzed by RT-quantitative PCR.
FIGURE 5.
FIGURE 5.
Pli1-related phenotypes in nup132Δ reverted by Pli1 expression. A, Western blots of SUMO conjugates or tubulin in the indicated strains in which Pli1 expression was induced (+), or not (−), from the nmt41 promoter for 24 h. The black arrowhead indicates the position of free mature SUMO. B, serial dilutions of the indicated strains transformed with empty control vector or pREP41-Pli1 that expresses Pli1 were spotted onto media without or with CPT grown at 35 °C. C and D, serial dilutions of the indicated strains were spotted onto rich (YES) or minimum selection (LAH) media at 32 °C, without or with the indicated drugs. YES, yeast extract, dextrose, amino acid supplement mixture; HU, hydroxyurea; CPT, camptothecin.
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