title: The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis.
abstract: RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
authors.forename: Sandesh
authors.initials: S
authors.lastname: Regmi
authors.forename: Karen G
authors.initials: KG
authors.lastname: Rothberg
authors.forename: James G
authors.initials: JG
authors.lastname: Hubbard
authors.forename: Larry
authors.initials: L
authors.lastname: Ruben
mesh_terms: D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D048749:Cytokinesis / Q000187:drug effects*; D016054:DNA, Protozoan / Q000235:genetics; D010442:Peptide Chain Initiation, Translational; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D016054:DNA, Protozoan; D020648:Peptide Elongation Factor 1; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000841:Anisomycin
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