JCS007955 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Structural comparisons among TbTLK1, TbTLK2 and Arabidopsis Tousled kinase. (A) A schematic representation of the different domains. The catalytic domains (CAT) are shown in black, the putative nuclear localization sequences (NLSs) in gray, and the predicted coiled-coil motifs indicated by striped boxes. Mw, molecular mass. (B) Alignments of the deduced amino acid sequences of TbTLK1, TbTLK2 and the Arabidopsis Tousled kinase. The positions of the NLS, the coiled-coil motifs and the CAT domains in TbTLK1 and TbTLK2 are indicated.

• Supplemental Figure S2 -

  Fig. S2. Overexpression of TbTLK1 and TbTLK2 in a TbAUK1 RNAi background (A) and effects of overexpressing the wild-type and the kinase-dead mutants of TbTLK1 and TbTLK2 on T. brucei growth and cell cycle progression (B,C). (A) Northern blot analysis. Total RNA from the non-induced and tetracycline-induced TbAUK1 RNAi cells overexpressing TbTLK1 or TbTLK2 were blotted onto a nitrocellulose membrane and probed with a TbAUK1 gene fragment to reveal expression of TbAUK1. α-Tubulin was included as sampling control. (B) The wild-type and kinase-dead mutants of TbTLK1 and TbTLK2, each tagged with a triple HA epitope at the C-terminus, were cloned into the pLew100 vector and transfected into 29-13 cells. Tetracycline was added to induce overexpression of the proteins, and cell growth was monitored by counting the cell numbers daily. (C) The control cells and cells after tetracycline induction for three days were fixed, stained with propidium iodide and examined by flow cytometry.

• Supplemental Figure S3 -

  Fig. S3. Endogenous tagging of TbTLK1 and TbTLK2 and subcellular localization of endogenous TbTLK1 and TbTLK2. (A) Schematic delineation (not to scale) of the overexpression construct and the endogenous tagging construct in the same cell line. Endogenous TbTLK1 and TbTLK2 were each tagged at the C-terminus with the HA epitope in the respective overexpressing cell lines using the integrating constructs pC-TbTLK1-HA-bla and pC-TbTLK2-HA-bla. The successful transfectants express both the HA-tagged endogenous TbTLK1 or TbTLK2 and the tetracycline-inducible HA-tagged overexpressed TbTLK1 or TbTLK2. (B) Western blot analysis. Cell lysates from the non-induced and tetracycline-induced cells were blotted with antibody against (‘α’) HA and antibody against α-tubulin (as the sampling control). Without tetracycline induction, the detected bands represent the tagged proteins at their endogenous levels, but, upon induction by tetracycline, the bands represent a summation of both the endogenous and the overexpressed proteins. This allows a direct comparison of the levels of overexpressed and endogenous proteins from the same cells. (C) Subcellular localization of endogenous HA-tagged TbTLK1 and TbTLK2. Non-induced cells, expressing the endogenous HA-tagged proteins, were fixed, stained with FITC-conjugated mAb against HA and counterstained with DAPI. Owing to the relatively low level of endogenous TbTLK2 protein, a much longer time of exposure was used to capture the fluorescent signal. The arrows point to the bright spots localized to the spindle poles. Bar, 2 μm.

• Supplemental Figure S4 -

  Fig. S4. The domains in TbTLK1 and TbTLK2 required for nuclear and spindle pole localization. The T. brucei cells expressing the HA-tagged chimeric proteins (see Fig. 3) were fixed with 4% paraformaldehyde, immunostained with FITC-conjugated mAb against HA and counterstained with DAPI. The arrows point to the bright spots localized to the spindle poles. Bar, 2 μm.

• Supplemental Figure S5 -

  Fig. S5. Expression of TbAUK1, TbTLK1 and TbTLK2 in yeast strains. Lysates from three yeast strains, which harbor the pGBK-TbAUK1-Myc/pGAD-TbAUK1-HA, pGBK-TbAUK1-Myc/pGAD-TbTLK1-HA and pGBK-TbAUK1-Myc/pGAD-TbTLK2-HA constructs, were prepared, separated by SDS-PAGE and stained with a mAb against Myc and a mAb against HA, respectively. A Coomassie-Blue staining of the gel served as a loading control. Note that TbTLK1 migrates slower than TbTLK2 protein by SDS-PAGE, suggesting that TbTLK1 is most likely also autophosphorylated.

• Supplemental Figure S6 -

  Fig. S6. Sequence alignments of TbAsf1A and TbAsf1B together with the Asf1 homologs from S. cerevisiae, Drosophila and human. The alignment shows the significant level of identity shared by both TbAsf1A and TbAsf1B in comparison with the Asf1 homologs from S. cerevisiae, Drosophila and human.