Generation of Tb<i>GAPDHL</i> and Tb<i>PGKL</i> procyclic null mutants.

<p>(<b>A</b>) Generation of Tb<i>GAPDHL</i> null mutants. (<b>B</b>) Generation of Tb<i>PGKL</i> null mutants. Cartoon schematics denote gene loci annotated with HindIII (H) restriction sites for (i) wild-type loci; (ii) loci following gene disruption and (iii) following endogenous gene-tagging with GFP (Tb<i>GAPDHL</i> only). Southern analysis of genomic DNA digested overnight at 37°C with HindIII shows blots probed sequentially with either coding sequence from the targeted gene (probe o) or sequence from the 3′ intergenic region (probe u). Relative positions of the probes are shown in the cartoon schematics. In (<b>A</b>) the order of lanes is 1, wild-type <i>GAPDHL</i>^+/+<i>T. brucei</i>; 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4-5, <i>GAPDHL</i>^+/− heterozygotes from lanes 2 and 3, respectively, in which endogenous tagging of the remaining wild-type allele results in expression of a recombinant e<i>GFP:GAPDHL</i>; 6, a <i>GAPDHL^−/−</i> mutant obtained from the stable transformation of the phleomycin-resistant heterozygote cells from lane 2. In (<b>B</b>) the order of lanes is 1, wild-type <i>PGKL</i>^+/+, 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4–5, independently obtained <i>PGKL</i>^−/− mutants derived from the stable transformation of heterozygous cell lines analyzed in lanes 2 and 3, respectively.</p

PFR localization of <i>Tb</i>GAPDHL.

<p>(<b>A</b>) Localization of GFP::<i>Tb</i>GAPDHL in procyclic <i>T. brucei</i> cells. (<b>B</b>) Indirect immunofluorescence of detergent-extracted cytoskeletons using the monoclonal antibody L8C4 to detect the major PFR protein PFR2 suggests GFP::<i>Tb</i>GAPDHL is a novel PFR component. Insets 1 and 2 indicate that at the proximal end of the flagellum PFR2 incorporation into flagellar skeleton does not begin prior to GFP::<i>Tb</i>GAPDHL incorporation – <i>cf</i> inset 2 in (<b>C</b>). (<b>C</b>) A lack of co-localization in indirect immunofluorescence of cytoskeletons using the monoclonal antibody L3B2 indicates GFP::<i>Tb</i>GAPDHL is not a cytoplasmic FAZ component: inset 1 indicates flagellar GFP::<i>Tb</i>GAPDHL fluorescence extends beyond the end of the cell body as denoted by L3B2 labelling of the cytoplasmic FAZ filament; inset 2 highlights how assembly the cytoplasmic FAZ filament detected by L3B2 initiates before assembly of GFP::<i>Tb</i>GAPDHL into the flagellar architecture. (<b>D</b>) Change in YFP::<i>Tb</i>GAPDHL localization in Tb<i>CaM</i> RNAi mutants: following RNAi induction and failure of PFR assembly YFP::<i>Tb</i>GAPDHL co-localizes with aggregates containing PFR2 protein (detected by indirect immunofluorescence with monoclonal antibody L8C4). (<b>E</b>) PFR localization of GFP::<i>Tb</i>GAPDHL is retained in <i>snl-2</i> RNAi mutants; detergent extracted cytoskeletons were also stained for indirect immunofluorescence with L13D6 to highlight failure to incorporate either PFR1 or PFR2, the two major PFR components, into the flagellar architecture. DIC, differential interference contrast; N, nucleus; K, kinetoplast. Scale bars denote 5 µm.</p

Flagellar localization of <i>Tb</i>PGKL.

<p>(<b>A</b>) Indirect immunofluorescence using monoclonal antibody BB2 reveals axonemal localization of Ty::<i>Tb</i>PGKL in detergent-extracted procyclic <i>T. brucei</i> cytoskeletons. Cytoskeletons were stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect mitochondrial (kinetoplast, K) and nuclear (N) DNA. The inset shows how the indirect immunofluorescence signal extends close to the kinetoplast, consistent with axoneme association. Scale bar denotes 5 µm. (<b>B</b>) Immunoblot analysis of detergent- and NaCl-extracted flagella isolated from procyclic cells expressing Ty::<i>Tb</i>PGK-like protein using BB2 detects a single band of the expected molecular mass.</p

Surface representation of TbGAPDHL with substrates.

<p>The representation is colored according to sequence conservation (blue-red, low to high) by Consurf. The catalytic Cys residue, neighboring residues (white sticks) and bound ligands (covalently attached glyceraldehyde-3-phosphate and NAD; ball and stick) from superimposed <i>G. stearothermophilus</i> GAPDH (PDB code 3cmc) are shown, with H-bonds illustrated as dotted lines. Corresponding residues in the TbGAPDHL model (purple sticks) are functionally incapable. Residues are labelled as template/model.</p

Sequence alignment of trypanosomatid GAPDHL proteins with authentic GAPDH.

<p>The alignment was built using MUSCLE and sequences are named with species abbreviations (Tb =  <i>T. brucei</i>, Tc =  <i>T. cruzi</i>, Lm =  <i>L. major</i>, Bs =  <i>B. saltans</i>, Gt =  <i>Geobacillus stearothermophilus</i>, Mb =  <i>Mycobacterium bovis</i>, Hs =  <i>Homo sapiens</i>, Ce =  <i>Caenorhabditis elegans</i>, Mj =  <i>Methanocaldococcus jannaschii</i>, Pt =  <i>Picrophilus torridus</i>) followed by a locus code (kinetoplastid sequences) or UniProt accession. For kinetoplastid sequences, cGAPDH indicates the cytosolic isoform and gGAPDH the glycosomal enzyme. Only one each of the tandem copies of gGAPDH is shown for each trypanosomatid. Residues mentioned in the text are highlighted as white on purple.</p

Range and mean percentage amino acid identities between GAPDH(-like) groups in trypanosomatids.

<p>Each group contains sequences (eliminating tandem duplicates) from <i>T. brucei, T. cruzi, T. vivax, L. braziliensis, L. mexicana, L. major, L. infantum, L. tarentolae</i>, and <i>Endotrypanum monterogeii</i>.</p