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The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

Steven Sykes, Anthony Szempruch, Stephen Hajduk
Steven Sykes
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA
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Anthony Szempruch
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA
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Stephen Hajduk
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA
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DOI: 10.1128/EC.00214-14
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  • FIG 1
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    FIG 1

    α-KDE1 is essential in BF T. brucei. Effect of α-KDE1 RNAi knockdown on the growth and morphology of BF T. brucei. (A) Growth of α-KDE1 RNAi T. brucei cells in culture at 37°C in the presence (+) or absence (−) of doxycycline. (B) Northern blot analysis of the levels of α-KDE1 and β-tubulin mRNAs. Total cell RNA was isolated following induction with doxycycline, fractionated on agarose gels, and hybridized with specific radioactively labeled probes for α-KDE1 and tubulin. (C) DIC images taken from videos of α-KDE1 RNAi T. brucei cells following induction with doxycycline for 0, 6, 12, and 18 h. The position of the expanding posterior vacuole is indicated (arrow). (D) SEM of α-KDE1 RNAi T. brucei following treatment with doxycycline. The position of the flagellum (f) is indicated.

  • FIG 2
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    FIG 2

    Localization of the α-KDE1 RNAi-induced vacuole. (A to C) Following induction with doxycycline for 6, 12, or 18 h, α-KDE1 RNAi T. brucei cells were incubated at 3°C with ConA-FITC, fixed, incubated with antibodies against the PFR protein, and stained with DAPI. The positions of the DAPI-stained kinetoplast (K) and nucleus (N) are indicated, as is that of bound ConA (arrow).

  • FIG 3
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    FIG 3

    α-KDE1 RNAi causes flagellar-pocket swelling. TEM images of α-KDE1 RNAi BF T. brucei are shown. (A) Low-magnification image of a field of cells 18 h after RNAi induction showing a high percentage of cells having a large intracellular vacuole. α-KDE1 RNAi-treated cells taken at the time of doxycycline induction (B) and after 6 h (C), 12 h (D), 18 h (E), and 24 h (F). The positions of the flagellar pocket (FP), flagellum (F), and kinetoplast (K) are indicated.

  • FIG 4
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    FIG 4

    Flagellar and plasma membranes are recruited to form the expanding flagellar pocket. TEM images of BF T. brucei following induction of α-KDE1 RNAi with doxycycline are shown. (A) Plasma membrane-associated pellicular microtubules are found on the membrane of the expanding flagellar pocket. The inset is a higher magnification of a portion of the flagellar membrane with associated subpellicular microtubules (SM). (B) Both axonemes (Ax) and the PFR protein, stripped of flagellar membrane, are displaced to the cytoplasm of α-KDE1 RNAi T. brucei cells. The inset is a higher-magnification view of a stripped axoneme and associated PFR protein in the cytoplasm.

  • FIG 5
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    FIG 5

    Morphological changes in glycosomes in α-KDE1 RNAi T. brucei. TEM images of BF T. brucei following induction of α-KDE1 RNAi with doxycycline are shown. (A to C) At 18 h after induction of α-KDE1, RNAi revealed clusters of elongated glycosomes throughout the cytoplasm but predominately near the flagellar pocket. The positions of the flagellar pocket (FP), glycosomes (G), kinetoplast (K), and nucleus (N) are indicated.

  • FIG 6
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    FIG 6

    Localization of α-KDE1 to the glycosome of BF T. brucei. α-KDE1 was tagged with a C-terminal HA epitope and used to prepare a constitutively expressing α-KDE1–HA cell line. (A) Total cell protein from wild-type (WT) and α-KDE1–HA (E1) cells was fractionated by SDS-PAGE and analyzed by Western blotting. On the left is the Coomassie blue-stained gel, and on the right is the blot following incubation with anti-HA antibody. The values to the left are molecular sizes in kilodaltons. (B, C) Localization of α-KDE1–HA by immunofluorescence microscopy relative to the mitochondrion stained with MitoTracker (B) and aldolase (C). The positions of the nucleus (N) and kinetoplast (K) are indicated.

  • FIG 7
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    FIG 7

    α-KDE1 contains an N-terminal glycosome-targeting signal. (A) Alignment of N-terminal amino acid sequences of α-KDE1, human and yeast peroxisomal, trypanosome glycosomal, and trypanosome mitochondrial proteins. The proposed trypanosome α-KDE1 MTS sequence (red) and PTS2 sequence (green) are shown. The arginine at position 4 and the leucine at position 5 (yellow) overlap in the predicted MTS and PTS2 sequences. Residues highly conserved in all PTS2 sequences are in bold (positions 4, 5, 8, and 12 in α-KDE1). (B) A fusion construct used to produce an α-KDE1–eGFP reporter contains the N-terminal 18 amino acids of α-KDE1 and the coding sequence for eGFP. (C, D) Following induction, eGFP localization was determined by fluorescence microscopy with cells stained with antialdolase antibody (C) and Mitotracker (D). The positions of the nucleus (n) and kinetoplast (k) are indicated.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 -

      Supplemental Figures S1 to S7 and additional experimental details.

      PDF, 424K

    • Supplemental file 2 -

      Supplemental Movie S1: motility of BF T. brucei is altered by α-KDE1RNAi (see Fig. S3). Video of cells by DIC at the time of induction (0 h).

      MOV, 643K

    • Supplemental file 3 -

      Supplemental Movie S2: motility of BF T. brucei is altered by α-KDE1RNAi (see Fig. S3). Video of cells by DIC at 6 h postinduction.

      MOV, 888K

    • Supplemental file 4 -

      Supplemental Movie S3: motility of BF T. brucei is altered by α-KDE1RNAi (see Fig. S3). Video of cells by DIC at 12 h postinduction.

      MOV, 946K

    • Supplemental file 5 -

      Supplemental Movie S4: motility of BF T. brucei is altered by α-KDE1RNAi (see Fig. S3). Video of cells by DIC at 18 h postinduction.

      MOV, 1.1M

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The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes
Steven Sykes, Anthony Szempruch, Stephen Hajduk
Eukaryotic Cell Mar 2015, 14 (3) 206-215; DOI: 10.1128/EC.00214-14

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The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes
Steven Sykes, Anthony Szempruch, Stephen Hajduk
Eukaryotic Cell Mar 2015, 14 (3) 206-215; DOI: 10.1128/EC.00214-14
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