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. 2002 Aug;13(8):2771-82.
doi: 10.1091/mbc.e02-03-0122.

Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome

Miguel Lafarga  1 , Maria Teresa Berciano, Emma Pena, Isabel Mayo, Jose G Castaño, Dirk Bohmann, João Pedro Rodrigues, João Paulo Tavanez, Maria Carmo-Fonseca
Affiliations

Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome

Miguel Lafarga et al. Mol Biol Cell. 2002 Aug.

Abstract

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin-proteasome pathway of proteolysis. We coined the term clastosome (from the Greek klastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place.

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Figures

Figure 1
Figure 1
Mammalian cell nuclei contain domains highly enriched in proteasomes. Antibodies directed against the 20S catalytic subunit of the proteasome were used to perform immunofluorescence on a variety of mammalian cell types. These included neurosecretory neurons of the supraoptic nucleus (A), astrocytes (B), and oligodendrocytes (C) obtained from squash preparations of rat hypothalamus, and primary cultures of Schwann cells prepared from rat sciatic nerve (D), human fibroblasts WI-38 (E), and HeLa cells (F). Although anti-20S proteasome antibodies label both nucleus and cytoplasm, staining of the nucleoplasm is more intense. Note the presence of brightly labeled intranuclear structures, of heterogeneous size and shape. Bar, 10 μm.
Figure 2
Figure 2
Ubiquitin and proteins degraded by the proteasome colocalize in proteasome-domains. Primary Schwann cells were double-labeled with antibodies directed against 20S proteasome and c-Jun (A and F), 19S proteasome and c-Jun (B and G), 20S proteasome and c-Fos (C and H), ubiquitin and c-fos (D and I), and ubiquitin and c-Jun (E and J). (K–O) Overlay of red and green images. Overlapping of red and green staining produces a yellowish color. Bar, 10 μm.
Figure 3
Figure 3
Proteasome-enriched domains disappear in the presence of proteasome inhibitors. HeLa cells were either mock treated (A) or incubated for 3 h in the presence of E64 (B), lactacystin (C), and MG132 (D). The cells depicted in E were allowed to recover for 12 h after treatment with MG132. All cells were immunolabeled with antibodies directed against the 20S catalytic core of the proteasome. Bar, 10 μm. (F) Proportion of cells containing proteasome-enriched domains and the number of bodies per cell were estimated. After treatment with E64, the proportion of cells that contain bodies does not significantly differ from the control population. The graph depicts means ± SEs (three separate experiments were performed and a total of 300 cells analyzed for each drug treatment).
Figure 4
Figure 4
Serum stimulation of fibroblasts induces the appearance of proteasome-enriched domains. Primary human fibroblasts (WI-38) grown in the presence of 0.1% fetal calf serum (FCS) for 4 d were stimulated for 75 min with 20% FCS. Immunofluorescence was performed using antibodies directed against 20S proteasome (A) or 19S proteasome (B). Cells were additionally double-labeled with anti-20S proteasome and anti-Hsp70/Hsc70 antibodies (C and D). Bar, 10 μm.
Figure 5
Figure 5
Stress induces the formation of proteasome-enriched domains in rat brain neurons. Hypotalamic neurosecretory neurons were obtained from control animals (A and B) and animals sacrificed 3 h after intraperitoneal injection of an hypertonic saline solution to induce osmotic stress (C–E). Cells were immunolabeled with antibodies directed against 20S proteasome (A and C) or 19S proteasome (B). Cells were additionally double-labeled with either anti-19S proteasome (D, red staining) and anti-c-Fos antibodies (D, green staining), or anti-19S proteasome (E, red staining) and anti-c-Jun antibodies (E, green staining). (D and E) Overlay of red and green images; colocalization produces a yellowish color. Bar, 10 μm. (F) Proportion of neurons containing proteasome-enriched domains and the number of bodies per cell were estimated at 0, 3, and 24 h after saline injection. The graph depicts means ± SEs (for each time point four animals were sacrificed, and from each animal ∼150 neurons were analyzed).
Figure 6
Figure 6
Adenovirus E1A oncoproteins localize in proteasome-enriched domains and stimulate their assembly. HeLa cells transiently transfected with plasmid pE1A, which expresses adenovirus E1A proteins, were double-labeled with anti-E1A antibody M73 (green staining) and either anti-19S proteasome (B and C, red staining) or anti-ubiquitin antibodies (E and F, red staining). (C and F) Overlay of red and green images; colocalization produces a yellowish color. Note that in some cases the E1A proteins occupy only a fraction of the ubiquitin-labeled structures (F, arrow). In E, the arrow indicates a domain enriched in ubiquitin-conjugates but devoid of E1A. Bar, 10 μm. (G) Proportion of transfected cells containing proteasome-enriched domains and the number of bodies per cell were estimated in a total of 300 randomly selected cells. The graph depicts means ± SEs.
Figure 7
Figure 7
Electron microscopy reveals that proteasome-domains correspond to complex nuclear bodies. Ultrathin sections from primary Schwann cells were immunogold-labeled with antibodies directed against 20S proteasomes (A, 5-nm gold particles), 19S proteasomes (B, 10-nm gold particles), ubiquitin (C, 10-nm gold particles), and c-Jun (D, 10-nm gold particles). Bar, 0.1 μm.
Figure 8
Figure 8
PML protein is present in clastosomes. HeLa cells were double-labeled with anti-PML antibody (green staining) and either anti-20S proteasome (A and C, red staining) or anti-ubiquitin antibodies (D and F, red staining). (C and F) Overlay of red and green images; colocalization produces a yellowish color. Note that PML occupies only a fraction of large clastosomes (C and F, arrow). The arrow in B points to a PML body that does not concentrate proteasomes. In D, the arrow points to a clastosome that contains little, if any, PML. Bar, 10 μm.
Figure 9
Figure 9
Overexpressed c-Jun protein forms aggregates that recruit proteasomes but are distinct from clastosomes. Ultrathin sections of HeLa cells transiently transfected with a plasmid encoding His6-tagged human c-Jun were immunogold-labeled with anti-20S proteasome (A and B) and anti-His6 antibodies (C). The HeLa cells were either mock treated (A) or incubated for 12 h in the presence of MG132 (B) or lactacystin (C). Bar, 0.3 μm. (D–I) HeLa cells were transiently transfected with a plasmid encoding His6-tagged human c-Jun (D and G), human c-Jun harboring a deletion of the δ domain (E and H), and chicken v-Jun (F and I). The cells were double-labeled with anti-His6 (D–F) and anti-20S proteasome antibodies (G–I). All three forms of the Jun protein produce aggregates in the nucleoplasm (D–F arrows); however, only c-Jun aggregates recruit proteasomes (G, arrows). Bar, 10 μm.
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