Ci 234 5 6 7 8 9

Fig. 3. Kinetics of accumulation of Bis, MT, and Hsp70 mRNAs in HeLa cells following exposure to copper. HeLa cells (lanes C, controls) were grown for 12 h in Dulbecco's modified minimal essential medium (DMEM) high glucose (Sigma) supplemented with 2 mM glutamine and 100 U/mL penicillin, 50 pg/mL streptomycin, and 10% fetal calf serum (Gibco). Before each experiment, the medium was replaced with DMEM containing 0.1% fetal calf serum. After 20 h, the cells were treated for different times (1 h, lanes 1 and 2; 2 h, lanes 3 and 4; 3 h, lanes 5 and 6; 5 h, lanes 7 and 8; 6 h, lanes 9 and 10) with 250 pM CuSO4 (lanes 1, 3, 5, 7, and 9) or 500 pM (lanes 2, 4, 6, 8, and 10). RNA was extracted with guanidine thiocyanate buffer and 10 pg of total RNA per sample were separated on a 1.5% agarose gel in the presence of formaldehyde and transferred to nylon membranes (Hybond N+, Amersham). The membranes were hybridized in stringent conditions to the following probes: Bis cDNA, nucleotide region 915-1728; hHsp 70 (40) (kind gift of Dr. Gabriella Santoro) and hMTI-e (41) full-size cDNAs. Levels of RNA were normalized for equal amounts of 28S RNA in each lane.

Fig. 3. Kinetics of accumulation of Bis, MT, and Hsp70 mRNAs in HeLa cells following exposure to copper. HeLa cells (lanes C, controls) were grown for 12 h in Dulbecco's modified minimal essential medium (DMEM) high glucose (Sigma) supplemented with 2 mM glutamine and 100 U/mL penicillin, 50 pg/mL streptomycin, and 10% fetal calf serum (Gibco). Before each experiment, the medium was replaced with DMEM containing 0.1% fetal calf serum. After 20 h, the cells were treated for different times (1 h, lanes 1 and 2; 2 h, lanes 3 and 4; 3 h, lanes 5 and 6; 5 h, lanes 7 and 8; 6 h, lanes 9 and 10) with 250 pM CuSO4 (lanes 1, 3, 5, 7, and 9) or 500 pM (lanes 2, 4, 6, 8, and 10). RNA was extracted with guanidine thiocyanate buffer and 10 pg of total RNA per sample were separated on a 1.5% agarose gel in the presence of formaldehyde and transferred to nylon membranes (Hybond N+, Amersham). The membranes were hybridized in stringent conditions to the following probes: Bis cDNA, nucleotide region 915-1728; hHsp 70 (40) (kind gift of Dr. Gabriella Santoro) and hMTI-e (41) full-size cDNAs. Levels of RNA were normalized for equal amounts of 28S RNA in each lane.

terminal tail of Bis protein and was used to detect the subcellular localization of Bis protein in control HeLa cells or in the same cells grown for different times in the presence of 250 pM CuSO4. In control cells, Bis protein appeared equally distributed in the cytoplasm (Fig. 4A). When HeLa cells were exposed to copper for 3 and 6 h Bis showed a perinuclear accumulation (Fig. 4B,C). Because Hsp70 has been shown to interact with the Bis protein, we next examined if the cellular localization of Hsp70 changed in cells exposed to copper. Using an antibody against the inducible form of Hsp70 (iHsp70), we observed that following exposure to the metal, the iHsp70 protein concentrated in the RER as well in a perinuclear compartment (Fig. 5B,C). These data are in agreement with the formation of complexes between iHsp70 and Bis in cadmium-exposed cells, demonstrated in immunopre-cipitation experiments (37).

2.4. Synthesis and Regulation of Bis Protein in HeLa Cells

The synthesis of the Bis protein was analyzed in vitro and in vivo. The full-length Bis cDNA was subcloned in pBluescript KS, transcribed in the corresponding mRNA, and translated in vitro in the presence of radioactive amino acids. The resulting in vitro translated polypeptide corresponded to 84 kDa, the expected molecular mass of the Bis protein (Fig. 6A, lane 1). We next examined if the same polypeptide was present in HeLa cells and copper regulated its synthesis. Cells were grown in presence or absence of 250 pM CuSO4 for 10 h and Western blot experiments were performed on the protein lysates, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/

Fig. 4. Copper-regulated cellular trafficking of Bis protein. HeLa cells were grown as described previously in the absence (A) or presence of 250 ||M CuSO4 for 3 h (B) and 6 h (C), plated on glass coverslips and fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, washed two times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were then treated with Bis antibody (dilution 1:400) for 20 min at room temperature, washed with PBS three times, and incubated with goat anti-rabbit FITC-conjugated IgG (dilution 1:100) (Sigma) for 20 min before observation under a fluorescence microscope.

Fig. 4. Copper-regulated cellular trafficking of Bis protein. HeLa cells were grown as described previously in the absence (A) or presence of 250 ||M CuSO4 for 3 h (B) and 6 h (C), plated on glass coverslips and fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, washed two times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were then treated with Bis antibody (dilution 1:400) for 20 min at room temperature, washed with PBS three times, and incubated with goat anti-rabbit FITC-conjugated IgG (dilution 1:100) (Sigma) for 20 min before observation under a fluorescence microscope.

Fig. 5. Localization of Hsp70 in cells exposed to copper. HeLa cells were grown as described previously in the absence (A) or presence of 250 |J,M CuSO4 for 3 h (B) and 6 h (C), plated on glass coverslips were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature, washed with PBS two times, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were treated for 20 min at room temperature with a mouse monoclonal antibody raised against the inducible form of Hsp70 (dilution 1:200), and after three washes with PBS, the cells were incubated for 20 min with a FITC-conjugated anti-mouse IgG antibody (dilution 1:100) (Sigma) before observation under a fluorescence microscope.

Fig. 5. Localization of Hsp70 in cells exposed to copper. HeLa cells were grown as described previously in the absence (A) or presence of 250 |J,M CuSO4 for 3 h (B) and 6 h (C), plated on glass coverslips were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature, washed with PBS two times, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were treated for 20 min at room temperature with a mouse monoclonal antibody raised against the inducible form of Hsp70 (dilution 1:200), and after three washes with PBS, the cells were incubated for 20 min with a FITC-conjugated anti-mouse IgG antibody (dilution 1:100) (Sigma) before observation under a fluorescence microscope.

Fig. 6. In vitro and in vivo synthesis of the Bis protein. (A) The plasmid (pBluescript KS), containing the full-length Bis cDNA, was linearized by digestion with XhoI and was transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system and T3 RNA polimerase (Promega), a mixture of amino acids minus methionine and 2 |L of 14.3 mCi/mL [35S] methionine (Amersham) according to the manufacturer's instructions. A reaction without the plasmid was also performed as a negative control. The translated product were reduced, alkylated, and separated on 10% SDS-PAGE gel (42) and revealed by autoradiography. (c) Negative control; 1, transcribed and translated Bis cDNA. (B) HeLa cells were grown as described previously in the absence (C) or presence of 250 |M CuSO4 for 10 h (lane 1). Total cell extracts were reduced, alkylated. and separated on 10% SDS-PAGE (42). Western blot was performed using the rabbit polyclonal anti-Bis antibody. Migration of the 14C-labeled protein molecular-weight markers are indicated on the left.

Fig. 6. In vitro and in vivo synthesis of the Bis protein. (A) The plasmid (pBluescript KS), containing the full-length Bis cDNA, was linearized by digestion with XhoI and was transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system and T3 RNA polimerase (Promega), a mixture of amino acids minus methionine and 2 |L of 14.3 mCi/mL [35S] methionine (Amersham) according to the manufacturer's instructions. A reaction without the plasmid was also performed as a negative control. The translated product were reduced, alkylated, and separated on 10% SDS-PAGE gel (42) and revealed by autoradiography. (c) Negative control; 1, transcribed and translated Bis cDNA. (B) HeLa cells were grown as described previously in the absence (C) or presence of 250 |M CuSO4 for 10 h (lane 1). Total cell extracts were reduced, alkylated. and separated on 10% SDS-PAGE (42). Western blot was performed using the rabbit polyclonal anti-Bis antibody. Migration of the 14C-labeled protein molecular-weight markers are indicated on the left.

PAGE), using our anti-Bis antibody. As shown in Fig. 6, the Bis protein was indeed present in control cells (Fig. 6B, lane C) and its synthesis was positively regulated by copper (Fig. 6B, lane 1).

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