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. 2010 Jun 29;5(6):e11371. doi: 10.1371/journal.pone.0011371

Figure 1. Vpr associates with proteasome in fission yeast and mammalian cells.

Figure 1

A. Vpr is displaced from the nuclear membrane by overproduction of Uch2. Fission yeast cells carrying or not carrying Uch2 were stained with DAPI 17 hrs after vpr gene induction. Green color, GFP; Blue color, nuclear DNA. B. Vpr is displaced from the nuclear membrane in the cut8 mutant. Mts4, a fission yeast homologue of mammalian S2, is a 19S proteasome-associated protein. Cut8 displays normal phenotype at the permissive 25°C, but shows mutant phenotype at non-permissive 37°C. C. Co-migration of Vpr with proteasome in fission yeast cells (i) and HeLa cells (ii) analyzed by glycerol gradient. Extracts from fission yeast cells expressing vpr were fractionated by centrifugation on a 10–40% glycerol gradient. Equal amounts of proteins from each fraction of the gradient were separated on 12% SDS-PAGE and probed with antibodies against Vpr and 19S (Mts4) subunits of the proteasome [34]. Lanes 1–8 indicates different fractions collected from the top (low molecular weight) to bottom of the gradient (high molecular weight). Note that not all fractions are shown here. D.i. Co-immunoprecipitation shows interaction of Vpr with Mts2 in yeast cells. IP was carried out with anti-HA as described previously [59]. A HA-tag alone plasmid control was used in this experiment. The recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-HA, anti-Vpr and anti-Mts2 antibodies. CL, cell lysates; IP:HA, immunoprecipitation with a HA-tagged control plasmid; IP:HA-Vpr, immunoprecipitation with a HA-Vpr carrying plasmid. ii. HeLa cells were transfected with HA-Vpr or HA-Kir2.1 (control). Kir2.1 is an irrelevant protein to Vpr and used here as a control. IP was carried out with anti-HA, recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-S2 (a mammalian homologue of fission yeast Mts4) and anti-S5a antibodies. iii. HeLa cells were co-transfected with pSG5-ZZ-β1, which codes for a proteasomal β1subunit [38], or control pSG5-ZZ plasmid (Ctr) together with HA-Vpr. The protein A-tagged β1 or control protein were pulled down by anti-protein A antibody, then blotted with anti-HA antibody.