Figure 1

mSigR1 abnormally accumulates in the ER and induces cellular toxicity. (a) Model of the homotrimeric SigR1 (based on ref. 16). Subunits are represented in gray, secondary structure of one subunit is color-coded; each monomer is composed of an amino-terminal transmembrane domain (red) which crosses the ER membrane from lumen to cytosol. The transmembrane domain is followed by two α-helices (green) that lead to a β-barrel (light violet), the putative ligand binding domain. Two carboxy-terminal α-helices (blue) form a hydrophobic membrane-embedded surface. The location of the critical residue Glu-102 (E102Q) is marked by a red dot. Red star and red triangle represent two recently discovered mutations.⁹ (b) Reticular pattern of SigR1 staining combined with a distinct nuclear envelope localization (lower panel) in wtSigR1-transfected MCF-7 cells; globular mSigR1 aggregates (arrows; see also enlargement, middle row) in MCF-7 cells expressing mSigR1. Immunofluorescence; scale bars, 15 μm. (c) Co-immunofluorescence of wtSigR1 and mSigR1 with emerin as a nuclear envelope marker in MCF-7 cells (arrowheads). Note that the focal emerin accumulations (arrows) co-localize with SigR1 aggregates. Scale bar, 10 μm. (d and e) Co-localization of wtSigR1 and mSigR1 with the ER markers KDEL and (e) calreticulin in MCF-7 cells. Scale bar, 15 μm. (f and g) Co-labelling of SigR1 with the β-cop (ER-Golgi-associated compartments) and GM130 (Golgi marker). Note the co-localization of mSigR1 with β-cop and the Golgi dispersal (arrowheads) in mSigR1 expressing MCF-7 cells. Scale bar, 10 μm. (h) Co-immunofluorescence of wtSigR1 and mSigR1 with mito-red as a marker for mitochondria in MCF-7 cells. Scale bar, 10 μm. (i) Quantification of the mito-red staining depicted in (h) showing numbers of enlarged mitochondria/random field of view. (j) NSC34 and MCF-7 cells were co-transfected either with empty pcDNA vector, wtSigR1 or mSigR1 together with the luciferase gene downstream of the ERSE promoter. After 48 h, luciferase units (AU) were analyzed as a measure of ER stress. *P<0.05,**P<0.005, #not significant. (k) MCF-7 cells were transfected with pcDNA, wtSigR1 or mSigR1 as described above and analyzed for ER stress and UPR induction by immunoblotting. (l) Quantification of the band intensities normalized with α-tubulin depicted in k. Values represent the mean±S.D. of three independent experiments. *P<0.05. (m) Ubiquitin immunoreactivity of wtSigR1 and mSigR1 in MCF-7 cells. Scale bar, 10 μm. (n) Co-localization of mSigR1 aggregates with the accumulated 20s subunit of the proteasome in MCF-7 cells expressing mSigR1. Scale bar, 10 μm. (o) Chymotrypsin-like proteasomal activity assays were performed using MCF-7 cells transfected with pcDNA, wtSigR1 and mSigR1, as described in material and methods. Note the significant decrease in proteasome activity due to mSigR1 expression when compared to wtSigR1 and pcDNA. Means±S.D. of three independent experiments each performed in triplicate. *P<0.05, **P<0.005, # not significant