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doi: 10.1242/10.1242/jcs.00123

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A developmentally regulated ARF-like 5 protein (ARL5), localized to nuclei and nucleoli, interacts with heterochromatin protein 1

¹ Institute of Molecular Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
² Institute of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

View larger version (59K): [in a new window]   Fig. 1. Alignment of deduced amino acid sequences of ARL5, ARLs and ARF1. Sources of sequences are: hARF1 (Bobak et al., 1989); hARL1 (Zhang et al., 1995); hARL2 (Clark et al., 1993); hARL3 (Cavenagh et al., 1994); hARL4 (Lin et al., 2000); hARL5 [this study (Smith et al., 1995)]; mARL5 (this study). Amino acids identical in at least five of the seven sequences compose the consensus sequence shown at the top. Underlines indicate consensus sequences for GTP binding and hydrolysis. Double underline indicates consensus sequences for nuclear localization signal. Black boxes indicate amino acids identical in hARL4, hARL5 and mARL5 sequences. Gray boxes indicate amino acids similar in hARL4, hARL5 and mARL5 sequences.

View larger version (83K): [in a new window]   Fig. 2. ARL5 mRNA in mouse tissue and embryos. Blots containing poly (A)+ RNA from adult mouse tissues (A) or whole embryos at different developmental stages (B) were hybridized with a random-primed, 32P-labeled mARL5 cDNA probe. Hybridization with a ß-actin probe was a control for sample loading.

View larger version (59K): [in a new window]   Fig. 3. Immunolocalization of endogenous hARL5 in hepatoma Hep3B and Huh7 cells. (A) Specificity of antibody against ARL5. 100 ng of the indicated purified recombinant His-tagged ARL (a,b) and the indicated amounts of purified His-tagged hARL5 (c) were subjected to SDS-PAGE in 12.5% gels. Positions of protein standards (32.9 and 24.8 kDa) are on the left. Proteins were transferred to nitrocellulose and reacted with (a) anti-Histag or (b,c) anti-ARL5 antibodies, followed by detection using the ECL system (A,B). (B) Subcellular distribution of hARL5. Nuclear (N), membrane (M) and cytosolic (C) fractions of Hepatoma Hep3B or Huh7 cells were prepared as described in Materials and Methods. Equivalent amounts (from total homogenate) of each fraction were analyzed by electrophoresis and immunoblotting using specific antibodies against ARL5, -tubulin (cytosolic marker), or PCNA (nuclear marker). (C) Immunolocalization of ARL5. Hepatoma Hep3B and Huh7 cells on glass coverslips, treated as described in Materials and Methods, were incubated with affinity-purified anti-hARL5-peptide (a,d) or ßCOP antibodies (c,f). Coverslips were mounted on Mowiol (supplemented with Hoechst 33258; b,e) and inspected with a Zeiss Axiophot equipped for epifluorescence. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 4. Transient expression of hARL5 and its mutants in COS-7 cells. (A) Diagrams of wild-type (WT) hARL5 and four hARL5 mutants with amino acid numbers below. Residues 188 to 201 contain the bipartite NLS of ARL5. ARL(dC) lacks 24 amino acids (177 to 201) at the C-terminus. Positions of the mutations Q80L and T35N, and MIR deletion (128-133) are also indicated. (B) COS-7 cells transfected with GFP fusion constructs of hARL5(WT), hARL5(Q80L), hARL5(T35N), or hARL5(dC) were grown on glass coverslips, fixed with formaldehyde, incubated with mouse anti-nucleolin (nucleolar marker), and detected with Alexa-488-conjugated anti-mouse IgG antibody. Coverslips were mounted on Mowiol (supplemented with Hoescht 33258) and inspected with a Zeiss Axiophot equipped for epifluorescence. Phase-contrast microscopy and GFP-hARL5 fluorescence (green) are shown. In the bottom two rows, arrows indicate nucleolins, and arrowheads indicate Golgi marker p58. Bar, 10 µm

View larger version (44K): [in a new window]   Fig. 5. Interaction of hARL5 with HP1 in the two-hybrid system. (A) Expression of LexA-ARL fusion proteins. Yeast reporter strain L40 was transformed with the indicated LexA construct, pBTM116 (LexA only), or pLexA-lamin. Samples (20 µg) of cell lysates were subjected to SDS-PAGE in a 10% gel, then stained with Coomassie blue (b, upper panel). Proteins were transferred to nitrocellulose and reacted with anti-LexA antibodies (a, upper panel; b, lower panel), or anti-ARL5 antibodies (a, lower panel) as indicated, followed by detection using the ECL system. (B) Interaction of hARL5 and mutants with HP1 in the two-hybrid system. Yeast reporter strain L40 co-transformed with pACT2-HP1 and the indicated pLexA-ARL construct or pLexA-lamin were plated on synthetic histidine-containing medium lacking leucine, tryptophan, uracil and lysine (His+ plate, upper panel). Colonies from His+ plates were assayed for ß-galactosidase activity by a filter assay to test for specificity (lower panel). Colonies from His+ plates were also patched on His- selective plates lacking histidine, leucine, tryptophan, uracil, and lysine (His- plate, middle panel). (C) Interaction of hARL5 and mutants with HP1, HP1ß, and HP1 in the two-hybrid system. Yeast reporter strain L40 co-transformed with pACT2-HP1, -HP1ß, or -HP1, and the indicated pLexA-ARL5 construct or pLexA-lamin were plated and assayed as described above (a). Proteins were transferred to nitrocellulose and reacted with anti-HA antibodies (b, lower panel), followed by detection using the ECL system. Samples (20 µg) of transformed yeast lysates were subjected to SDS-PAGE in a 10% gel, then stained with Coomassie blue (upper panel b). Proteins were transferred to nitrocellulose and reacted with anti-HA antibodies (b, lower panel), followed by detection using the ECL system. Positions of protein standards (kDa) are on the left.

View larger version (43K): [in a new window]   Fig. 6. Interaction of hARL5 and mutants with four HP1 deletion derivatives in the two-hybrid system. The diagram of HP1 and its deletion derivatives was adapted from Seeler et al., 1998 (Seeler et al., 1998) (A, left). The adapted figure was reproduced with permission from National Academy of Sciences, USA. Yeast reporter strain L40 co-transformed with pACT2-HP1 deletion derivatives and the indicated pLexA-ARL5 construct or pLexA-lamin were plated and assayed as described above (B). Panel A (right) shows the summary of the interaction result. Samples (20 µg) of transformed yeast lysates were subjected to SDS-PAGE in a 10% gel (C, upper panel). Positions of protein standards (kDa) are on the left. Proteins were transferred to nitrocellulose and reacted with anti-HA antibodies (C, lower panel), followed by detection using the ECL system. Red underlines (A) indicate regions of HP1 that interact with ARL5.

View larger version (44K): [in a new window]   Fig. 7. In vitro interaction of hARL5 with HP1 and importin-. (A) In vitro interaction of GST-HP1 with hARL5 constructs. hARL5, hARL5(Q80L), hARL5(T35N), hARL5(dC), or hARL5(dMIR) were synthesized by in vitro translation. Samples (5 µl) were mixed with 10 µg of GST or GST-HP1 immobilized on glutathione-Sepharose beads and incubated at 4°C for 1 hour before beads were washed four times with 1 ml of the same buffer. Bound proteins were eluted by boiling in 20 µl of 2x protein sample buffer and separated by SDS-PAGE in 12% gel. Input lane contained 10% of the amount added to beads. Proteins were stained with Coomassie Blue to ensure equal loading, and the bound proteins were visualized by autoradiography. (B) In vitro interaction of GST-importin- with hARL5 constructs. 10 µg of GST or GST-importin- immobilized on glutathione-Sepharose beads were mixed with 5 µl of in vitro translated proteins of interest as described as above.

View larger version (62K): [in a new window]   Fig. 8. Transient expression of hARL5 or its mutants and HP1 in COS-7 cells. COS-7 cells co-transfected with Flag-tagged HP1- and GFP-tagged constructs of hARL5, hARL5(Q80L), hARL5(T35N), hARL5(dC), hARL5(dMIR), or hARL5(Q80LdMIR) were grown on glass coverslips, fixed with formaldehyde, and incubated with mouse anti-Flag (d,h,l,p,t,x) or mouse anti-nucleolin (nucleolar marker) antibodies. Coverslips were mounted on Mowiol (supplemented with Hoechst 33258; b,f,j,n,r,v) and inspected with a Zeiss Axiophot equipped for epifluorescence. Phase-contrast microscopy (a,e,i,m, q,u) and GFP-hARL5 fluorescence (c,g,k,o,s,w; green) are shown. Nucleolins are indicated by arrows. Co-localization of HP1- and hARL5(Q80L) is indicated by arrowheads. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 9. In vivo interaction of hARL5 with HP1. For immunoprecipitation of the HP1-hARL5 complex, COS-7 cells were co-transfected with an hARL5 construct and HP1. After 48 hours, cells were harvested, washed three times in PBS, suspended in PBS, and placed on ice. DSP was then added. After 1 hour at 0°C, the reaction was stopped. The cells were harvested, washed and lysed. After centrifugation (10,000 g, 15 minutes) of lysates, M2 anti-Flag affinity gel was added to the supernatants, followed by incubation at 4°C for 3 hours with agitation. Beads were then washed three times with TBS and dispersed in appropriate amounts of sample buffer. Eluted proteins were subjected to western blotting analysis using anti-GFP polyclonal antibody (lower panel). Samples of total cell lysates were also subjected to western blotting analysis using anti-Flag and anti-ARL5N antibodies (upper two panels).

View larger version (36K): [in a new window]   Fig. 10. Myristoylation of hARL5. (A) Recombinant hARL5, hARL1, and yARL3 were synthesized in E. coli co-expressing yeast N-myristoyltransferase that were grown in medium containing (3H)-myristic acid. Samples of bacterial proteins (20 µg) were subjected to SDS-PAGE in a 15% gel, which was fixed in 10% acetic acid and 45% methanol for 30 minutes, incubated in Amplify (Amersham) for 20 minutes, dried, and exposed to Hyper-film (Amersham) for 41 hours at -80°C. (B) COS-7 cells were transfected with pcDNA3.1A C-terminal Myc-tagged ARL5(WT) or ARL5(G2A) and metabolically labeled with [3H]myristic acid before immunoprecipitation of cell extracts with anti-Myc antibody. Beads were dispersed in 20 µl of 2x SDS sample buffer and extracted proteins analyzed by SDS-PAGE in 12% gel. Gels were fixed, dried and exposed to Hyperfilm for 21 days at -80°C (upper panel). Part of each immunoprecipitated sample was subjected to western blotting analysis using anti-Myc (middle panel) and anti-ARL5N antibodies (lower panels). Asterisks indicate the lower bands of both ARL5(WT) and ARL5(G2A).