Analysis of Lsg1p protein family
Database similarity searches
The translated sequence of the Homo sapiens gene FLJ11301 (GenBank accession no. NP_060855) was used to search the non-redundant protein database at the National Center for Biotechnology Information using the PSI-BLASTP program (15). Homologues were identified in Homo sapiens (GenBank accession identifier BAA92116), Mus musculus (XP_148574), Danio rerio (AAH66695), Caenorhabditis elegans (NP_490904), Caenorhabditis briggsae (CAE74467), Drosophila melanogaster (NP_569915), Anopheles gambiae (EAA13064), Saccharomyces cerevisiae (NP_011416), Schizosaccaromyces pombe (NP_593948), Arabidopsis thaliana (NP_172317), Zea mays (AAD41267), Encephalitozoon cuniculi (CAD26329), Eremothecium gossypii (NP_985506) and Plasmodium falciparum (NP_702181). The sequence corresponding to Rattus norvegicus had to be reconstructed using an insertion from Mus musculus, probably owing to an incorrect gene prediction (XP_213604).
Phylogenetic analysis
The 14 orthologous sequences were aligned using the ClustalW program [16]. PSI-BLAST searches on the NCBI protein database were performed using different representatives of the YRG family as seed, according to the bibliography, and were iterated until members of the closest subfamily were found in the list of hits. The sets of orthologous sequences were manually checked for sequence integrity and to clarify subfamily definitions. Progressively larger multiple sequence alignments were built by constructing multiple sequence alignments of each subfamily, which were manually polished and added together stepwise. At each step, the parts outside the central GTPase domain, which often showed no homology across subfamilies (and therefore should not be aligned), were trimmed to facilitate the production of the next multiple sequence alignment. The final multiple sequence alignment was used to produce the corresponding phylogenetic tree (excluding the non-aligned regions) using ClustalW. The full list of sequences used for the tree and their database identifiers are given as supplementary material [see Additional file 1].
Cell culture, transfections, immunostaining and fluorescence microscopy
HeLa (ATCC CCL-2) and Vero (ATCC CCL-81) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and penicillin/streptomycin at 37°C in an atmosphere of 5% CO2. Cells were seeded on to glass coverslips, Nunc plates or LabTek dishes and were transfected using Fugene6 (Roche) according to the manufacturer's protocols. For immunocytochemistry, transiently transfected HeLa cells were grown on coverslips and fixed in ice-cold methanol for 5 min at -20°C. The cells were then washed again and incubated in PBS for 20 min. Primary and secondary antibodies were diluted in PBS. The cells were incubated with primary antibodies followed by secondary antibodies for intervals of 30 min with three washing steps in between. The coverslips were then mounted in Mowiol on glass slides. Images of the stained cells were acquired using either a Zeiss Cell Observer System or a Leica AOBS confocal laser-scanning microscope.
GTP binding and GTPase activity measurements
Nucleotide binding was measured by the filtration method. Recombinant proteins were incubated in 20 mM Tris-HCl pH 7.5, 1 mM DTT, 5 mM MgCl2, 10 mM EDTA, 0.5 g/l bovine serum albumin, (3H)GTP or (3H)GDP (7,7 Ci/mmol, Amersham-Pharmacia-Biotech) and cold 30 μM GTP or GDP. After incubation at 30°C for the indicated times, samples were diluted in 500 μl of ice-cold washing buffer (20 mM Tris-HCl pH 7.5, 25 mM MgCl2 and 100 mM NaCl) and applied to a nitrocellulose filter (0.45 μm, Millipore). The filters were rinsed with 4 × 4ml ice-cold washing buffer and the radioactivity retained on the filters was determined by scintillation counting.
GTPase activity measurement by HPLC was described by Ahmadian et al. 1999 [17].
siRNAs transfection and western blotting
siRNA sequences were BLAST searched against the human genome to ensure that they were specific for hLsg1. The hLsg1 siRNA sequence showed no exact or near exact matches to any other sequence in the human genome and are therefore hLsg1-specific. siRNAs were synthesized by EUROGENTEC. hLsg1 siRNA (5'-UGGAGAGAAACUGCAAGACTT-3') targets nucleotides 506–524 of human hLsg1 relative to the first nucleotide of the start codon.
Cells were seeded into 12-well plates. Twenty-four hours later, they were transfected with 1.68 μg of siRNA per well (unless otherwise noted). Transfections were as described [28] with the following modifications. Additional OptiMEM (Invitrogen) was not added, and medium was removed before transfection and replaced with 400 μl of OptiMEM. Full-serum medium (unless otherwise noted) was added 4 h post-transfection. At the indicated times post-transfection, the cells were washed twice with PBS and detached from the plate with PBS EDTA. Whole cell extract was obtained by lysing the cells with RIPA buffer containing protease inhibitors and DTT. Protein concentrations were measured using the Bradford assay. Extracts were run on 8% polyacrylamide gels (12% for actin) and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin), then probed with either rabbit polyclonal anti-hLsg1 or anti-actin (Santa Cruz Biotechnology) antibody for 1 h at room temperature (overnight at 4°C for actin), washed, and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Signals were detected using the ECL-Plus reagent (Amersham Biosciences).
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