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. 2001 Jan 30;98(3):1012-7.
doi: 10.1073/pnas.98.3.1012. Epub 2001 Jan 23.

Proteins associated with the promyelocytic leukemia gene product (PML)-containing nuclear body move to the nucleolus upon inhibition of proteasome-dependent protein degradation

K Mattsson  1 , K Pokrovskaja, C Kiss, G Klein, L Szekely
Affiliations

Proteins associated with the promyelocytic leukemia gene product (PML)-containing nuclear body move to the nucleolus upon inhibition of proteasome-dependent protein degradation

K Mattsson et al. Proc Natl Acad Sci U S A. .

Abstract

Several recent findings have indicated that the promyelocytic leukemia gene product (PML) oncogenic domains (PODs) are involved in proteasome-mediated degradation of ubiquitinated proteins. We wanted to examine the intracellular distribution of PML protein in the presence of a proteasome inhibitor. We used high-resolution microscopy to study the distribution of PML protein and other POD-associated proteins along with the proteasomes themselves under normal conditions and in cells treated with the proteasome inhibitor, MG132. Inhibition of the proteasomes in MCF-7, HeLa, and IB-4 cell lines resulted in a radical redistribution of the POD-associated proteins PML, Sp100, and SUMO-1. After 6-10 h of MG132 treatment, PML, Sp100, and SUMO-1 were no longer detectable in the PODs and accumulated mainly in the nucleolus. Moreover, MG132 treatment changed the cellular distribution of the proteasomes. Interestingly, this included the accumulation in euchromatin areas of the nucleus and within the nucleoli. Several non-POD-associated proteins did not change their cellular distribution under the same conditions. The accumulation of POD-associated proteins and proteasomes in the nucleoli of MG132-treated cells indicates that these proteins may target the nucleoli under normal conditions and that the nucleolus may have a function in the regulation of proteasomal protein degradation.

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Figures

Figure 1
Figure 1
Subnuclear distribution of PML after DMSO or MG132 treatment of MCF-7, HeLa, or LCL IB-4 cell lines. Combination of DNA (blue) and PML (green) is shown in the left panels (a, d, g, j, m, p), middle panels show PML alone (b, e, h, k, n, q), combination of phase-contrast and immunofluorescence of PML is shown in right panels (c, f, i, l, o, r). IB-4 cells cultured in the presence of DMSO (a–c) with PML distributed throughout the nucleoplasm excluding the nucleoli. Cells cultured with 5 μM MG132 for 6 h (d–f) show staining of PML inside the nucleoli (d, f) in addition to some nuclear dots. HeLa cells cultured in the presence of DMSO (g–i) or with 5 μM MG132 for 15 h (j–l). PML accumulation upon MG132 treatment is shown in one of three nucleoli (l). MCF-7 cells cultured in the presence of DMSO (m–o) or with 5 μM MG132 for 15 h (p–r). PML accumulates in the nucleoli of MG132-treated cells (r).
Figure 2
Figure 2
Nucleolar localization of PML in MG132-treated MCF-7 cells. High magnification image of double staining for fibrillarin and PML or for B23 and PML. (a) Phase-contrast field combined with fibrillarin staining (red). (b) Phase-contrast and nucleolar PML (green) staining representing the same field as shown in a. (c) Overlap between fibrillarin and PML staining. (d) Phase-contrast field combined with nucleolar B23 staining (red). Panels e [phase-contrast and nucleolar PML staining (green)] and f overlap between B23 and PML.
Figure 3
Figure 3
Sp100 changes subcellular distribution and disassociates from PML upon MG132 treatment of MCF-7 cells. High magnification of Sp100 (green) and PML (red) double-staining of DMSO (a–c) or MG132-treated MCF-7 cells (d–f). (c and f) Overlap of Sp100 and PML. In DMSO-treated cells, Sp100 colocalized to a large extent with PML (c). After MG132 treatment, Sp100 completely changed its localization (d and f); it accumulated in the nucleoli along with PML but without any colocalization (f). DNA staining in blue.
Figure 4
Figure 4
(A) Double staining of PML (green) and proteasomes (red) in DMSO- or MG132-treated MCF-7 cells. In DMSO-treated cells, proteasomes were homogeneously distributed throughout the nucleus excluding nucleoli (b). Double staining showed no obvious colocalization between the two proteins (c). MG132 treatment changed the nuclear distribution of both PML and proteasomes; they both accumulated in the nucleoli as shown in panel (d–f). (B) Subnuclear localization of proteasomes in MG132-treated MCF-7 cells. Proteasomes (green) accumulate in the euchromatin areas and nucleoli and avoid peripheral (solid arrowheads) and perinucleolar (concave arrowheads) heterochromatin. (C) a–c represent double staining for SUMO-1 (green) and PML (red) in DMSO-treated MCF-7 cells. Overlap image shows complete colocalization of the two proteins (c). Upon MG132 treatment, SUMO-1 and PML accumulated in the nucleoli (d and e) without colocalization. DNA staining in blue.
Figure 5
Figure 5
Three-dimensional stereoscopic reconstitution of PML (red) and SUMO-1 (green) double-stained cell (MCF-7) treated with MG132 showing that although both proteins accumulated in the nucleolus, they do not colocalize with each other any longer. The mathematically deblurred image was generated from a series of 11 optical sections, 0.3 μm apart. DNA staining in blue.
Figure 6
Figure 6
Effect of cycloheximide on the nucleolar accumulation of PML and proteasomes. (A) MCF-7 cells that were treated with MG132 alone (a and b) or with MG132 plus cycloheximide (c and d) over night did not change the nucleolar accumulation of PML (green). (B) The nucleolar accumulation of proteasomes (green) was not altered by the inhibition of protein synthesis in MCF-7 cells (a–d). DNA staining in blue.
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