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. 2008 Nov;70(3):724-45.
doi: 10.1111/j.1365-2958.2008.06443.x. Epub 2008 Sep 10.

The RACK1 Signal Anchor Protein From Trypanosoma Brucei Associates With Eukaryotic Elongation Factor 1A: A Role for Translational Control in Cytokinesis

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Free PMC article

The RACK1 Signal Anchor Protein From Trypanosoma Brucei Associates With Eukaryotic Elongation Factor 1A: A Role for Translational Control in Cytokinesis

Sandesh Regmi et al. Mol Microbiol. .
Free PMC article

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.

Figures

Fig. 1
Fig. 1
The identification of TAP-TbRACK1 binding partners. A. Expression of TAP-TbRACK1 and its distribution in cultured PF. A cloned PF cell line was produced that constitutively expressed the TAP-Tag fused to the amino terminus of TbRACK1. Cells were fractionated by centrifugation and each fraction was suspended in the original homogenate volume. Proteins were separated on a 12% polyacrylamide gel and analysed by Western blot with rabbit anti-TbRACK1 to evaluate the relative abundance of TAP-TbRACK1 (55.5 kDa) compared with endogenous TbRACK1 (34 kDa). B. Affinity purification of TAP-TbRACK1 and its associated proteins. In the left panel, Western blot with rabbit anti-TbRACK1 is used to identify TAP-TbRACK1 and endogenous TbRACK1 during the affinity purification steps. The TEV-protease eluted proteins from the IgG-Sepharose beads, while the Ca2+ wash and pooled EGTA eluates were from the calmodulin-Sepharose beads. A new band at 40 kDa was generated by the TEV-protease cleavage of the TAP-TbRACK1. The right panel is the Coomassie-stained gel of the EGTA eluate. The three predominant bands were excised and each band was analysed by LC MS/MS. Proteins that comprised each band are listed. C. TbRACK1, TAP-TbRACK1 and eEF1A cosediment on sucrose gradients. About 109 log phase cells were disrupted with glass beads followed by the addition of 1.2% (v/v) Triton X-100. The 14 000 g supernatant was applied to a 12 ml linear sucrose gradient (15–50%) and centrifuged for 2.5 h at 170 000 g (avg). Fractions (0.5 ml each) were collected and proteins were precipitated with methanol/chloroform. Each fraction was separated by SDS-PAGE on 12% acrylamide gels, and probed with rabbit anti-TbRACK1 (upper panel) or with mouse anti-eEF1A (lower panel).
Fig. 2
Fig. 2
TbRACK1 and eEF1A are binding partners. A. TbRACK1 associates with eEF1A in cell homogenates. Upper panel: Wild-type PF (AnTat1.1) were gently lysed, and the 10 000 g supernatant fraction was used for immunoprecipitation pull-downs. Each tube was incubated with combinations of cell homogenates, rabbit anti-TbRACK1 and protein A beads, as indicated. Western blot analysis was with mouse anti-eEF1A. In each panel, the input is shown in the left lane. Middle panel: 29-13 PF cells were transformed with AU1-eEF1A in pLEW100. After 3 days induction with 1 μg ml−1 tetracycline, cell homogenates were precipitated with anti-AU1 beads and the pull-down of TbRACK1 was monitored. Lower panel: Homogenates of parental 29-13 cells were precipitated with anti-AU1 beads and the pull-down of TbRACK1 was monitored. B. Purified native eEF1A and (His)6-TbRACK1 coimmunoprecipitate. Reactions contained different combinations of purified native eEF1A (10 μg) and recombinant (His)6-TbRACK1 (8 μg), as indicated. Anti-TbRACK1 pulled down eEF1A only when (His)6-TbRACK1 was present in the assay (upper panel). Ni-NTA pulled down eEF1A only when (His)6-TbRACK1 was present in the assay (middle panel). Anti-eEF1A pulled down (His)6-TbRACK1 only when eEF1A was present in the assay (lower panel). The antibody heavy chain (hc) is visible in the pull-down with anti-eEF1A (lower panel). The input is shown in the left lanes. C. Purification of native eEF1A from trypanosome homogenates. Coomassie-stained gel of the eEF1A purification. Individual lanes contain the 100 K supernatant; DE-52 flow through; calmodulin-Sepharose flow through; final wash; and EGTA eluate from the calmodulin-Sepharose column. Adjacent panel is the Western blot of the final eluate.
Fig. 3
Fig. 3
Bacterially expressed recombinant eEF1A does not interact with TbRACK1. A. eEF1A and TbRACK1 fail to interact by bacterial two-hybrid. BactrioMatch II validation reporter competent cells (Stratagene) were co-transformed with bait and prey, and then plated on non-selective M9+ plates. Ten colonies from the M9+ non-selective plates were suspended in liquid culture and re-plated onto either: M9+ medium; M9+ with 5 mM 3-AT; or M9+ with 5 mM 3-AT and 12.5 μg ml−1 streptomycin. For the sake of space, only four colonies are shown. Control plasmids were purchased from Stratagene, and consisted of bait (pBT-LGF2) and prey (pTRG-GAL1 1P). B. Bacterially expressed recombinant (His)6-eEF1A does not interact with recombinant (His)6-TbRACK1. Binding assays contained combinations of 8 μg (His)6-TbRACK1 and 8 μg (His)6-eEF1A, as indicated. Immunoprecipitation was with anti-TbRACK1 and proteins in the pull-down were detected with anti-His6 antibodies. eEF1A is not pulled down by these procedures. The input is in the left lane.
Fig. 4
Fig. 4
TbRACK1 is a component of trypanosome monosomes, and can be released with high concentrations of NaCl or 0.2% deoxycholate. A. Purification of monosomes from wild-type PF. Monosomes were partially purified through a step gradient of 20%/40% sucrose, and separated on a linear gradient of 5–25% sucrose. The OD254 was continuously recorded and 0.5 ml fractions were collected. Proteins in each fraction were precipitated by methanol/chloroform and separated by SDS-PAGE. Western blot was used to detect TbRACK1, TcP0 or α-tubulin. B. The crude ribosome fraction was incubated with 0.7 M NaCl for 30 min prior to loading on the sucrose gradient. C. The crude ribosome fraction was incubated with 0.2% deoxycholate for 45 min prior to loading on the sucrose gradient.
Fig. 5
Fig. 5
Identification of TbRACK1 in polysome preparations from PF. A. Polysomes were harvested from a 15–50% linear sucrose gradient. The OD254 was continuously recorded and 0.5 ml fractions were collected. Proteins in each fraction were precipitated by methanol/chloroform and separated by SDS-PAGE. Western blot was used to detect TbRACK1, TcP0 or α-tubulin. B. Polysomes were incubated with 0.5 mg ml−1 RNase A for 30 min prior to separation on the 15–50% linear sucrose gradient.
Fig. 6
Fig. 6
Polysomes from Trypanosoma cruzi epimastigotes contain TcRACK1. A. Antibodies detect RACK1 and P0 from T. brucei PF and T. cruzi epimastigotes with high specificity. Whole-cell homogenates (15 μg) were separated by SDS-PAGE and were analysed by Western blot with antibodies against TbRACK1 or TcP0. B. Polysomes were harvested from a 15–50% linear sucrose gradient. The OD254 was continuously recorded and 0.5 ml fractions were collected. Proteins in each fraction were precipitated by methanol/chloroform and separated by SDS-PAGE. Western blot was used to detect TcRACK1, TcP0 or α-tubulin in each fraction. C. Polysomes were incubated with 0.5 mg ml−1 RNase A for 30 min prior to separation on the 15–50% linear sucrose gradient.
Fig. 7
Fig. 7
Conditional knockdown of TbRACK1 changes the polysome profile and increases phosphorylation of a ribosomal protein in T. brucei PF. A. Polysome profiles were obtained from 1 × 109 TbRACK1 RNAi cells, either without induction of RNAi (−Tet), or after induction of RNAi (+Tet) for a 4 day period. Compared with uninduced cells and control wild-type cells, polysomes from TbRACK1-depleted cells had an increased peak height of the 80S peak relative to the polysomes and a decreased average length of the polysome profile. B. The effect of TbRACK1 RNAi on phosphorylation of a 30 kDa ribosomal protein. After 4 days of RNAi induction, the level of TbRACK1 decreased in whole-cell homogenates, as determined by Western blot (left panels). Purified ribosomes from these cells were also depleted of TbRACK1 (right panels). The level of phosphorylated threonine in a 30 kDa protein increased. TcP0 was used as a loading control. Molecular weight markers in kDa are shown for the phospho-threonine blot.
Fig. 8
Fig. 8
TbRACK1 RNAi cells are hypersensitive to anisomycin. A. Anisomycin affects growth of the TbRACK1 RNAi cells. A three-times cloned TbRACK1 RNAi cell line and the 29-13 parental cell line were grown for a 4 day period either without tetracycline or with 1 μg ml−1 tetracycline, as indicated. Increasing concentrations of anisomycin were added to the medium during the final 48 h. Cell density was recorded at the end of the 4 day period, and the value without anisomycin was set at 100%. Without anisomycin, the parental 29-13 cells grew to a density of 2.2 ± 0.08 × 107 cells ml−1, the RNAi cells without tetracycline grew to 1.9 ± 0.2 × 107 cells ml−1 while the RNAi cells with tetracycline grew to a density of 0.7 ± 0.01 × 107 cells ml−1. Each point is the average ± SE of two independent experiments. B. Phenotypic changes in trypanosomes after treatment with subtoxic levels of anisomycin. Cells were treated with combinations of 1 μg ml−1 tetracycline for a total of 4 days and 4 μg ml−1 anisomysin was added during the final 48 h, as indicated. The cells were viewed by DIC microscopy and at least 200 cells were scored for partial cleavage furrow ingression. Each bar is the average ± SE of two independent experiments. C. Phenotypes of cells treated with subtoxic levels of anisomycin. Cells from the experiment in (B) were fixed, permeabilized and stained with the long wavelength laser dye TOTO (DNA) and with rabbit anti-PFR (flagella). (a) 4 μg ml−1 anisomycin alone; (b) TbRACK1 RNAi alone; (c) TbRACK1 RNAi with 4 μg ml−1 anisomycin (low magnification). (d)–(e) are higher magnification of the TbRACK1 RNAi cells after treatment with 4 μg ml−1 anisomycin. In each panel, the bar is 15 μm.
Fig. 9
Fig. 9
TbRACK1 depletion works synergistically with a subtoxic dose of anisomycin to block cleavage furrow ingression during cytokinesis. A. Anisomycin at 4 μg ml−1 does not affect cell cycle progression of parental 29-13 cells or the TbRACK1 RNAi cells without induction of RNAi (−Tet). The cells were treated for 24 h with anisomycin, fixed with paraformaldehyde, stained with DAPI and scored for nuclei (N) and kinetoplasts (K). The majority of cells are 1N1K. Shown is the average ± SE for two independent experiments. B. The effects of 4 μg ml−1 anisomycin on cell cycle progression after depletion of TbRACK1 with RNAi (+Tet). Early time points after treatment with anisomysin (12 h or 24 h). The subtoxic dose of anisomycin caused cells with 2N2K and > 2N to accumulate within 12 h of treatment. C. The cytokinesis defect begins with an abnormal configuration of kinetoplasts and nuclei. Cells were fixed with paraformaldehyde, permeabilized and stained with TOTO (DNA) and rabbit anti-PFR (flagella). The 2N2K cells were evaluated as a population. The cells were postmitotic and 14% of them had not initiated cytokinesis (sum of the upper two panels) while 64% of the cells had a partial cleavage furrow (sum of the lower two panels). Cells were classified as having a wild-type configuration (KNKN; in which the pattern of kinetoplasts and nuclei is described starting from the posterior end of the cell), misaligned nuclei and kinetoplasts (KKNN), partial cleavage ingression (2N2K ingression) and advanced cleavage ingression (NKKN ingression). The number in parentheses is the percentage of the total 2N2K population represented by a specific phenotype.
Fig. 10
Fig. 10
Translational inhibition and cytokinesis. A. Anisomycin does not lower the levels of TbRACK1 in control cells or TbRACK1 depleted cells. Cell cultures were treated with combinations of tetracycline (1 μg ml−1) for 4 days and with anisomycin (4 μg ml−1 for 24 h), as indicated. Whole-cell homogenates (10 μg per lane) were separated by SDS-PAGE, and the expression levels of TbRACK1 or TcP0 were determined by densitometry of Western blots. A longer exposure was required to detect TbRACK1 in the RNAi cells +Tet (right panels). Blots similar to this one were quantified by densitometry and the level of TbRACK1 in each sample was expressed as the ratio of IDV values for TbRACK1:TcP0. B. A model to explain the synergy between anisomycin and depletion of TbRACK1. We propose that in wild-type cells (WT), translation is sufficient to sustain cell viability and cytokinesis. When expression of TbRACK1 is decreased with RNAi, the expression level of a cytokinesis protein approaches a critical threshold, while bulk translation continues. The translational inhibitor anisomycin at 4 μg ml−1 has no effect on cell cycle progression of control cultures, but we propose that it lowers expression of the cytokinesis protein below a critical threshold.

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