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Figure 1 hLsg1 is member of a large circularly permuted GTPase family. (A) Schematic representation of hLsg1 – hLsg1 encodes a protein of 658 amino acids comprising a central MMR/HSR1 GTPase domain (black box, 151–500), a coiled-coil domain (Hatched box, 126–151) and a nuclear localization signal (NLS, grey box, 638–654). Domain organization of the GTPase is indicated as well as the insertion (white box inside the black box, 220–320) separating the G4 motif from the G1 motif. (B) Phylogenetic tree of the YRG superfamily. We constructed a multiple alignment of representative sequences from the YqeH, YjeQ, EngA, and YlqF families. The alignment was produced using ClustalW followed by manual editing [16]. The tree was generated from the alignment using MrBayes v3 [45] (100000 generations with parameter n chains = 4; convergence occurred after 33600 generations; the tree is the consensus of 664 trees computed using MrBayes. No molecular clock was assumed and therefore the branch lengths have no meaning. The numbers indicate the fraction of trees displaying the grouping given by the branch). The root of the tree is the one given by the MrBayes output. (C) Distribution of YRG members in cellular compartments in different organisms.
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Figure 2 Nucleotide binding and GTPase activity of hLsg1. (A) GTP binding of hLsg1. Nucleotide binding was measured as described in Materials and Methods. BSA was used as control, while Sar1p-WT and the GDP-restricted Sar1p mutant (Sar1p-T39N) were used as positive and negative GTP binding controls, respectively. The graph is the sum of three separate experiments. (B) GTPase activity of purified recombinant and immunoprecipitated hLsg1. Elution times of GDP and GTP standards are indicated (top panel). GTPase activities of purified recombinant Sar1-WT and hLsg1 are shown (middle panel) as well as GTPase activities of immunoprecipitated Sar1p and hLsg1 (lower panel). Incubation times were identical (18 h) except for the hLsg1 precipitate (4 h). (C) Hydrolysis of GTP by hLsg1. GTPase activities of purified recombinant hLsg1 were analyzed by HPLC as described in Materials and Methods. A solution containing 5 μM hLsg1 and 200 μM GTP was incubated at 37°C. Samples were taken at different time-points and analyzed for percentages of GTP and GDP.
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Figure 3 hLsg1 is an essential protein. (A) Rapid disappearance of hLsg1 in cells transfected with hLsg1 specific siRNA. HeLa cells were transfected with an hLsg1-specific siRNA (+) or with a scrambled siRNA as a negative control (-), and were harvested at 0, 12 and 24 h post-transfection. Extracts were prepared and 30 μg of each sample were separated on an 8% polyacrylamide gel and analyzed for hLsg1 by western blotting. Untreated cell lysate (30 μg) from confluent HeLa cells was run as a control (Co). To assess the specificity of the siRNAs, 30 μg of each extract was run on a 12% SDS-PAGE gel and analyzed for actin content by western blotting. (B) Cell count. HeLa cells plated on coverslips and transfected with no siRNA (black box), hLsg1-specific siRNA (white box), or a scrambled siRNA (grey box) were fixed at 0, 12, 24 and 36 h post-transfection. Cells were labeled with Dapi and anti-hLsg1 antibodies and the cell number was counted. The graph is the sum of three independent experiments (C) YRG family member lethality. Literature survey and database searches indicating that YRG family members are essential.
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Figure 4 Subcellular localization analysis of hLsg1. (A) Subcellular localization of YFP and CFP tagged hLsg1 – HeLa cells were transiently transfected with hLsg1-YFP (1, 2), or CFP-hLsg1 (3) and visualized by fluorescence microscopy. (B) Localization of endogenous hLsg1. hLsg1 was visualized by staining with anti-hLsg1 antibodies, followed by Alexa 488-conjugated anti-rabbit antibodies. HeLa cells were transiently transfected with YFP-tagged lamin or Clontech ER-YFP marker. Coilin was visualized by monoclonal mouse anti-coilin antibodies, followed by rhodamine-conjugated anti-mouse antibodies. Bars = 10 μm
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Figure 5 hLsg1 localized to the nucleus upon Leptomycin B treatment. (A) Subcellular localization of YFP tagged hLsg1 mutants – HeLa cells were transiently transfected with YFP-hLsg1-1-600 (1) or YFP-hLsg1-480-658 (2), or co-transfected with YFP-hLsg1-480-658 and SRP19-MRFP (3), and visualized by fluorescence microscopy. (B) Localization of hLsg1 and mutants upon Leptomycin B treatment. HeLa cells were transiently transfected with hLsg1-YFP (a), YFP-hLsg1-1-600 (b), or YFP-hLsg1-480-658 (c), treated with 15 nM Leptomycin B, fixed at 0 h, 3 h and 5 h, and visualized by fluorescence microscopy.
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