Dislocation of HMG₃₅₀-HA and Insig-1-Myc involves p97/VCP. On day 1, HEK293 cells were transfected with a plasmid mixture consisting of 3 μg of pIRESneo2-HMG₃₅₀-HA, 0.5 μg of pcDNA3.1-Insig-1-Myc, and 0.5 μg of wild-type or K524A mutant of pcDNA3.1-p97-FLAG, as indicated. Total plasmid concentration was adjusted to 4 μg/dish with empty pcDNA3 vector. Forty-eight hours later, the cells were switched to medium B for additional 16 h after which they were treated for 2 h with ALLN (65 μM) with (+) or without (−) sterols. Cells were permeabilized, and supernatant (60 μg of protein) and pellet (20 μg of protein) fractions were analyzed by immunoblotting with the indicated antibodies. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively.

Sterol-stimulated formation and dislocation of HMGR · Insig-1-Myc complex. Sterol-depleted cells that express HMG₃₅₀-HA together (+) or without (−) Insig-1-Myc were pulse-labeled for 30min with of ³⁵S–cell-labeling reagent (100 μCi). Cells were chased in the presence of ALLN with (+) or without (−) sterols, permeabilized, and fractionated. (A) Aliquots of supernatant and solubilized pellet (5% of samples) were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (B) The rest 95% of the samples were first subjected to immunoprecipitation (first IP) of Insig-1-Myc with a mouse monoclonal anti-myc antibody, and immune complexes were analyzed by SDS-PAGE and IB with rabbit antibodies for the presence of Insig-1-Myc (ii) and coprecipitated HMG₃₅₀-HA (i). (C) The supernatants from the first IP were cleared with protein A-Sepharose beads and reprecipitated (second IP) with a mouse monoclonal anti-HA antibody and immune complexes were analyzed by SDS-PAGE for the presence of HMG₃₅₀-HA by IB with a rabbit anti-HA antibody (i) and by direct autoradiography (ii). Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively. Asterisks (Aii, lanes 5 and 10) denote a protein in the supernatant fraction that cross-reacted with the anti-myc antibody. (D) Densitometric analysis of the blots. The HA immunoreactive signal in Bi (including trailing smear) was divided by the Myc-immunoreactive signal in Bii (including trailing smear), setting the ratio at time zero as one. Results are the mean of two independent experiments. Closed symbols, sterol-depleted cells; open symbols, sterol-treated cells. (E) Sterol-depleted naïve or Insig-1-Myc–expressing cells were incubated for 2 h with ALLN and sterols, as indicated. Cells were permeabilized in solution C and fractionated. Proteins from the supernatant and pellet fractions were IP with rabbit polyclonal anti-myc antibodies. The immune complexes were resolved and IB with a mouse anti-HMGR monoclonal antibody (i; arrow) and then reprobed with mouse anti-myc monoclonal antibodies (ii). Aliquots of the fractions, representing 5% of protein input, were immunoblotted with rabbit anti-calnexin antibody.

Interaction between HMG₃₅₀-HA and Insig-1-Myc requires their expression in the same cell. Sterol-depleted cells expressing either HMG₃₅₀-HA, Insig-1-Myc, or both, and a mixture of cells expressing either proteins, were incubated for 2h with ALLN with (+) or without (−) sterols, and cells were permeabilized and fractionated. (A) Aliquots of supernatant (5%) and solubilized pellet (10%) (see Note) were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. The rest 95% of the supernatant fraction was divided into two equal portions. (B) One portion was directly immunoprecipitated (IP) with an anti-myc monoclonal antibody and immune complexes were analyzed by SDS-PAGE and immunoblotting with rabbit anti-myc and anti-HA antibodies, for the presence of Insig-1-Myc (ii) and coprecipitated HMG₃₅₀-HA (i), respectively. (C) To the other supernatant portion, NP-40 and NaDOC were added to 1% each, and the samples were immunoprecipitated and immunoblotted as described in B. Note: Because the supernatant was divided into two portions, the 5% sample of the initial fraction actually represents 10% of total protein input in each portion used for the analyses in B and C.

Ubiquitination, degradation, and dislocation of HMG₃₅₀-HA, HMGR and Insig-1-Myc. (A) Sterol-depleted cells, stably expressing either HMG₃₅₀-HA or Insig-1-Myc, were incubated for 2 h with ALLN (65 μM) with (+) or without (−) sterols (2 μg/ml 25-hydroxycholesterol plus 20 μg/ml cholesterol). Cells were lysed in solution D, and proteins from the postnuclear supernatant were immunoprecipitated (IP) with rabbit polyclonal anti-HA (lanes 1 and 2) or anti-myc (lanes 3 and 4) antibodies. The immune complexes were resolved by 5–15% SDS-PAGE, electroblotted onto nitrocellulose, and the membrane was first probed (IB) with a mouse anti-ubiquitin monoclonal antibody (top) and then reprobed with mouse anti-HA and anti-myc monoclonal antibodies (bottom). Arrowheads indicate the migration of the immunoprecipitated proteins. M_r markers (in kilodaltons) are shown. (B) Sterol-depleted cells, as in A, were pulse labeled for 30 min with ³⁵S-protein label mix (100 μCi/dish; 300 μCi/ml) and chased for the indicated time in unlabeled medium in the absence or presence of sterols. Cells were lysed in solution D, and proteins were immunoprecipitated with the mouse monoclonal anti-HA (top) or anti-myc (bottom) antibodies. The immune complexes were resolved by SDS-PAGE followed by fluorography. Densitometric analysis of the gels is shown. •○, HMG₃₅₀-HA; ■□, Insig-1-Myc; closed symbols, sterol-depleted cells; open symbols, sterol-treated cells. (C) Cells expressing both HMG₃₅₀-HA and Insig-1-Myc, were incubated for the indicated time with ALLN with (+) or without (−) sterols, as described in A. Cells were permeabilized in solution C and fractionated by centrifugation, as described under Materials and Methods. Aliquots of the supernatant (60 μg of protein) and solubilized membranes (20 μg of protein) were IB and reprobed with the indicated antibodies. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively. The asterisk (*) denotes a protein in the supernatant fraction that cross-reacted with the anti-myc antibody. M_r markers (in kilodaltons) are shown. (D) Densitometric analysis of experiments, as in C, of cells expressing only HMG₃₅₀-HA (light gray bars; n = 12), or cells expressing both HMG₃₅₀-HA and Insig-1-Myc or Insig-1-Myc alone probed with anti-HA (dark gray bars; n = 11) or with anti-Myc (black bars; n = 8) antibodies. The results (mean ± SEM) were evaluated by Student's t test, and p values are indicated; n.s., not significant. (E) On day 1, SRD-15 cells were transfected with 2 μg of pIRESneo2-HMG₃₅₀-HA along with 0.5 μg of an empty vector (−) or pcDNA3.1-Insig-1-Myc (+), as indicated. On day 2, the cells were replated in triplicate dishes in medium lacking sterols but containing 50 μM lovastatin and 50 μM MVA. On day 3, the cells were harvested immediately (0) or incubated for 2 h with 65 μM ALLN in the presence (+) or absence (−) of sterols. Cells were permeabilized, and supernatant (45 μg of protein) and pellet (22 μg of protein) fractions were analyzed by immunoblotting with the indicated antibodies. Asterisks denote a protein in the supernatant fraction that cross-reacted with the anti-myc antibody. (F) Sterol-depleted cells expressing both HMG₃₅₀-HA and Insig-1-Myc, were harvested immediately (0) or incubated for 2 h with 65 μM ALLN with (+) or without (−) sterols. Cells were permeabilized and fractionated by centrifugation, as described under Materials and Methods. The 20,000 × g supernatant was further centrifuged at 100,000 × g for 30 min. Samples of the pellet (100 μg of protein) and supernatant (30 μg of protein) fractions were analyzed by SDS-PAGE and IB with the indicated antibodies. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively. The asterisk (*) denotes a protein in the supernatant fraction that cross-reacted with the anti-myc antibody.

Cytosolic PNGase removes N-glycan from dislocated HMG₃₅₀-HA. (A) Mock-transfected cells or cells expressing HMG₃₅₀-HA were incubated for 2 h with ALLN (65 μM) with (+) or without (−) sterols. Cells were permeabilized in solution C and fractionated (lanes 1–8), or solubilized in solution D (lanes 9–14). Aliquots of supernatant (75 μg of protein), solubilized pellet (20 μg of protein), or whole cell lysates (120 μg protein) were treated (+) or mock treated (−) with 1000 U of EndoH and analyzed. (B) Cells were preincubated for 1 h with the indicated concentrations of Z-VAD-fmk before addition of ALLN and sterols. The cells were incubated for additional 2 h and then permeabilized and fractionated. Aliquots of supernatant (60 μg of protein) and solubilized pellet (20 μg of protein) were analyzed. (C) Cells were incubated with ALLN and sterols, in the absence (−) or presence (+) of Z-VAD-fmk (50 μM). At the indicated time points, cells were permeabilized and fractionated and aliquots of supernatant (75 μg of protein) and solubilized pellet (20 μg of protein) were analyzed. All samples were resolved by 5–15% SDS-PAGE and immunoblotted with anti-HA monoclonal antibody. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively.

Interactions with Insig-1-Myc and dislocation of HMG₃₅₀-HA mutants. Cells expressing the indicated constructs of HMG₃₅₀-HA, together (+) or without (−) Insig-1-Myc, were incubated for 2h with ALLN with (+) or without (−) sterols, permeabilized in solution C and fractionated. (A) Aliquots of supernatant and solubilized pellet (10% of samples) were analyzed by SDS-PAGE and IB with the indicated antibodies. (B) The rest 90% of the samples were subjected to IP with an anti-myc monoclonal antibody, and immune complexes were analyzed by SDS-PAGE and immunoblotting (IB) with rabbit anti-myc and anti-HA antibodies for the presence of Insig-1-Myc (ii) and coprecipitated HMG₃₅₀-HA (i), respectively. Closed and open arrowheads indicate glycosylated and un/deglycosylated forms of HMG₃₅₀-HA, respectively. M_r markers (in kilodaltons) are shown.