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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02452

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HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation

Department of Clinical and Experimental Medicine, University of Perugia, Policlinico Monteluce, 06122 Perugia, Italy

View larger version (50K): [in a new window]   Fig. 1. The Hops gene. (A) Northern blot analysis of Hops mRNA extracted from rat at different times following PH (h PH). Normal liver (NL) was used as a control. rRNA of the same samples were used as a loading control. (B) Protein sequence of HOPS. The ubiquitin-like domain (102-175) is underlined; the proline-rich domain (176-183) is in bold; the putative NES is boxed. (C) Schematic representation of HOPS. The characteristic domains of the amino acid sequence are indicated. (D) Western blot analysis of HOPS during liver regeneration in mouse using a specific antibody. NL and liver after PH (minutes and hours PH). Anti-CREB was used as a loading control.

View larger version (109K): [in a new window]   Fig. 2. Immunohistological analysis of HOPS in normal (NL) and regenerating liver at different times following PH (30 minutes to 72 hours) using (left column) anti-HOPS (original magnification x400 and inset, higher magnification, x1000). (Middle column) DAPI staining of the same slide. (Right column) Merged images of anti-HOPS and DAPI. Scale bars: 20 µm.

View larger version (68K): [in a new window]   Fig. 3. HOPS expression in H-35 hepatoma cells. (A) Western blot analysis of HOPS in (Top) H-35 hepatoma cells arrested by double thymidine excess. Time 0: the cell at the end of treatment; 2-12 h: hours after addition of serum and release from cell cycle arrest. (Bottom) Western blot analysis of HOPS at different times after serum deprivation in H-35 hepatoma cells. 0-72 h: hours of serum deprivation; +1, +2 h: hours after addition of serum to cells. (B) An aliquot of H-35 cells was treated with propidium iodide and assayed by FACScan analysis to evaluate the cell cycle. (Top) H-35 cells not treated; (middle) time 0: H-35 cells following treatment with double thymidine; (bottom) 72 sd: H-35 cells starving for 72 hours following serum deprivation (sd). (C) Immunolocalization of HOPS in H-35 cells at different times (12-72 hours) after serum deprivation (12-72 hours) and 2 hours after addition of serum (+2 hours). Scale bars: 20 µm.

View larger version (120K): [in a new window]   Fig. 4. HOPS localization in liver following cAMP induction. (Left colum) Localization of HOPS (anti-HOPS) in normal liver and at 15, 30, 60, 90 and 120 minutes following intraperitoneal injection of cAMP. (Middle column) DAPI staining of the same slides at different times following cAMP injection. (Right column) Merged images of anti-HOPS and DAPI. Scale bars: 20 µm.

View larger version (41K): [in a new window]   Fig. 5. Effect of leptomycin B (LMB) on HOPS shuttling. (A) Comparison of NES sequence in HOPS and other proteins. (B) Immunohistological staining (anti-HOPS) of H-35 hepatoma cells left untreated (–LMB) or treated with LMB (+LMB) resulted in accumulation of HOPS in nucleus in treated cells. DAPI: DAPI staining of the same slides; merge: merged images of anti-HOPS and DAPI. (C) Quantification of HOPS staining in the nucleus and cytoplasm in the presence or absence of LMB. Data are representative of five separate experiments. Standard errors of the mean are indicated by error bars. Scale bars: 20 µm.

View larger version (36K): [in a new window]   Fig. 6. Isolation of HOPS binding protein by the yeast two-hybrid system. HOPS full-length protein was used as bait. (A) Growth of transformants co-expressing HOPS and eEF-1A on selective medium. HOPS, HOPS-Gal4-DBD; eEF-1A, the clone isolated from the screening of the library; Gal4DBD, VP-16-AD and lamin are negative controls. Individual Trp+ and Leu+ transformants were plated on selective medium (Trp–Leu–) with histidine and adenosine (HIS+/ADE+) or without histidine and adenosine (HIS–/ADE–). (B) ß-galactosidase assay in selected colonies of yeast expressing HOPS and eEF-1A. ß-galactosidase units are expressed in Miller units. (C) Coimmunoprecipitation of HOPS-EF-1A complex from H-35 hepatoma cells. (Top) Western blot analysis of H-35 protein extract immuno-precipitated with preimmune and anti-HOPS sera (IP). H-35, protein extract as control. Detection was performed with anti-EF-1A. (Bottom) Western blot with anti-eIF-2a as control.

View larger version (27K): [in a new window]   Fig. 7. Inhibition of translation by HOPS in vitro and reduction of proliferation in vivo. (A) Luciferase cDNA was translated in an in vitro translation system with and without (C: control) different concentrations of purified recombinant HOPS protein (60-420 nM). Similar experiments were performed with or without purified recombinant GST protein. eEF-1A expression was analyzed by western blotting on reticulate lysates with different HOPS concentrations. (B) The synthesized luciferase was quantified by densitometric analysis after gel electrophoresis. The average results from three experiments are shown (in arbitrary units). Black bars: the amount of luciferase after the addition of different concentrations of HOPS; gray bars: the amount of luciferase after the addition of different concentrations of GST. (C) Percentage thymidine incorporation by stable H-35 stable clones (C1-C3) overexpressing HOPS with respect to H-35 control. (D) Number of puro selected NIH-3T3 cells at different days after infection. The gray line shows the number of control cells infected with plasmid Puro; the black line shows the number of cells infected with plasmid Puro containing HOPS.

View larger version (26K): [in a new window]   Fig. 8. Schematic representation of HOPS function. Cell proliferation signals and factors that increased cAMP allow export of HOPS via CRM-1. HOPS binds eEF-1A in the cytoplasm and then returns to the nucleus.

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