Article Figures & Data
Figures
- Figure 1. Bioinformatics-based identification and features of putative trypanosomatid Pex3 proteins.
(A) The secondary structure-based homology program HHpred was used to search a database of T. brucei gambiense with the known HsPex3 sequence. The uncharacterized target (T) protein XP_011780297.1 encoded by the Tbg972.11.11520 gene was identified with greater than 95% probability as having a similar predicted secondary structure (ss pred) as the query (Q), HsPex3. HHpred output includes a prediction of helices (H, h) and coils (C, c) (capitals represent a more reliable prediction), of conserved consensus residues (capitals represent a more reliable prediction) calculated from the query (Q) or Target (T), and a strength of alignment of amino acids according to their biophysical properties (“|” very good, “+” good, “·” neutral, “‒” bad, and “=” very bad) (Söding et al, 2005). (B) Alignment of the putative trypanosomatid Pex3 sequences from Leishmania major (Lm; Accession no. XP_001686984.1) and T. brucei (Tb; Accession no. XP_829090.1) with known Pex3 sequences from human (Hs; Accession no. NP_003621.1), the plant A. thaliana (At; Accession no. NP_001154410.1), the yeast S. cerevisiae (Sc; Accession no. NP_010616.3), the amoeba P. fungivorum (Pf; Accession no. PRP89031.1), and the stramenopile N. gaditana (Ng; Accession no. EWM25801.1). Sequences were aligned using Kalign(https://www.ebi.ac.uk/Tools/msa/kalign/). An amino acid that is identical to its corresponding amino acid in TbPex3 is highlighted in blue. An amino acid that is similar to its corresponding amino acid in TbPex3 is highlighted in yellow. Groupings of similar amino acids are as follows: (G, A, S), (A, V), (V, I, L, M), (I, L, M, F, Y, W), (K, R, H), (D, E, Q, N), and (S, T, Q, N). Dashes represent gaps. The conserved aromatic residues W/F of Pex3 required for interaction with Pex19 are shown in red and boxed. Other conserved residues that were mutated to evaluate the effect on the interaction between TbPex3 and TbPex19 in comparison with the interaction between ScPex3 and ScPex19 are boxed. See also Fig 2.
- Figure 2. TbPex3 displays an orthodox mode of binding to TbPex19.
(A) Yeast two-hybrid analysis of protein interactions between TbPex3 and TbPex19, and between ScPex3 and ScPex19. S. cerevisiae HF7c cells expressing Gal4-AD and Gal4-BD protein fusions to wild-type and point mutants of Pex3 and wild-type Pex19 were grown in liquid synthetic dropout medium and adjusted to an OD600 of 1.0. A 10-fold serial dilution series was spotted onto −Leu −Trp (left) and −His −Leu −Trp (right) plates. Growth on −Leu −Trp medium requires cells to have both AD and BD plasmids and is indicative of cell number. Growth on −His −Leu −Trp medium occurs only when there is a protein–protein interaction. (B) TbPex3 binds TbPex19. GST alone or GST-TbPex19 fusions immobilized on glutathione-sepharose beads were incubated with extracts of E. coli expressing either MBP-TbPex3, MBP-TbPex3-F102A, or MBP-TbPex3-L105A. Bound protein was detected by immunoblotting with anti-MBP antibody (upper panel, left). Total GST fusion proteins were visualized by immunoblotting with anti-GST antibody (lower panel, left). Panels at right show 5% of load. Numbers at left denote migration of molecular mass markers.
- Figure 3. TbPex3 is a glycosomal protein.
(A) TbPex3 is a glycosomal protein. PCF and BSF cells expressing TbPex3 C-terminally tagged with the HA epitope were fixed with formaldehyde and processed for immunofluorescence microscopy with mouse anti-HA antibodies and Alexa Fluor 488 rabbit antimouse IgG (top panels, green) and with rabbit anti-aldolase antibodies and Alexa Fluor 568 goat antirabbit IgG (middle panels, red). Merged images are presented in bottom panels. Bar, 2 μm. (B) TbPex3 cofractionates with glycosomal enzymes. Postnuclear lysates of PCF cells expressing Pex3-HA were fractionated on a discontinuous sucrose gradient. Equivolume fractions collected from the bottom of the gradient were analyzed by immunoblotting with antibodies to HA, aldolase, GAPDH, lipoamide dehydrogenase, and BiP.
- Figure 4. TbPex3 is essential for glycosome biogenesis and trypanosome viability.
(A) RNAi eliminates the TbPex3 transcript. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of transcript levels for TbPex3 and Tubulin (control) from uninduced (−Tet) and RNAi-induced (+Tet) PCF and BSF cells of T. brucei on day 3 of culture. (B) TbPex3-RNAi leads to mislocalization of the glycosomal matrix enzyme aldolase to the cytosol. Uninduced (−Tet) and RNAi-induced (+Tet) PCF and BSF cells of T. brucei on day 3 of treatment were processed for immunofluorescence microscopy with rabbit anti-aldolase antibodies and Alexa Fluor 488 goat antirabbit IgG. Bar, 2 μm. (C) TbPex3-RNAi–treated cells release glycosomal matrix enzymes at lower amounts of the detergent, digitonin, than do uninduced cells. Aliquots of untreated (−Tet) and RNAi-treated (+Tet) T. brucei PCF cells were incubated with increasing concentrations of digitonin and subjected to centrifugation. Supernatants were analyzed for the presence of aldolase, GAPDH, and tubulin. (D) Transmission EM of TbPex3-RNAi cells. Uninduced (−Tet) and RNAi-induced (+Tet) PCF cells and PSF cells of T. brucei were visualized on day 3 of treatment. Red arrowheads point to glycosomes. Bar, 5 μm. Quantification of EM images is presented in Table 1. (E) Pex3 is essential for cell viability of T. brucei. Growth curves of uninduced (−Tet) and TbPex3-RNAi–induced (+Tet) PCF cells and BSF cells. Error bars present SEM of triplicate readings.
- Figure S1. Time course of TbPex3-RNAi induction.
(A) Progressive reduction of the TbPex3 transcript with time of RNAi induction. Semiquantitative RT-PCR analysis of transcript levels for TbPex3 and Tubulin (control) from uninduced BSF cells (−Tet) and RNAi-induced BSF cells (+Tet) at 4, 6, and 8 h of induction. (B) TbPex3-RNAi leads to increasing numbers of aberrant glycosome structures, glycosome fragmentation, and mislocalization of the glycosomal matrix enzyme aldolase to the cytosol with time of RNAi incubation. Uninduced BSF cells (−Tet) and TbPex3-RNAi–induced BSF cells induced for 4, 6, and 8 h were processed for immunofluorescence microscopy with rabbit anti-aldolase antibodies and Alexa Fluor 488 goat antirabbit IgG. Bar, 5 μm.
Tables
- Table 1.
Glycosome number and average area in uninduced cells and TbPex3-RNAi–induced cells.
Form (number of cells analyzed) Glycosome number/cell volume (μm3) Glycosome average area ± SEM (μm2) PCF uninduced (n = 99) 0.078 0.46 ± 0.15 PCF induced (n = 99) 0.006 0.68 ± 0.36 BSF uninduced (n = 103) 0.078 0.78 ± 0.26 BSF induced (n = 103) 0.011 1.05 ± 0.31
