Figure 1.
Glycerolipid biosynthetic pathways in Leishmania.
AGAT, 1-acyl-glycerol-3-phosphate acyltransferase; ADR, alkyl/acyl-DHAP reductase; LmADS, alkyl-DHAP synthase; DHAP, dihydroxyacetonephosphate; LmDAT, DHAP acyltransferase; LmFAR, fatty acyl-CoA reductase; G3P, glycerol-3-phosphate; LmGAT, G3P acyltransferase; PA, phosphatidic acid. Genes encoding ADR and AGAT in Leishmania are unknown.
Figure 2.
Characterization of mutant forms of LmDAT.
(A) Schematic representation of human DHAPAT (hDHAPAT) and mutant forms of LmDAT. The grey rectangle, the black rectangle and the hatched area depict the HV tag, the conserved domain, and the C-terminal glycosomal targeting tripeptide, respectively, and the asterisk depicts the point mutation. B) DHAPAT activity was quantified as described in Materials and Methods. Equivalent of 0.5 mg protein extracts were applied for the assay. Null mutant alone or expressing HV-tagged wild-type and mutant forms of LmDAT were used as a source of protein extracts. Activity is expressed as percentage of the positive control, the wild type (WT). The assay was performed twice in duplicate, and the graph depicts one representative experiment. Standard deviations are shown. (C) Western blot analyses in the presence of V5-specific (upper; V5) and hypoxanthine guanine phosphoribosyltransferase specific (lower; HGPRT; loading control) antibodies. Equivalent of 5×107 cells were loaded in each lane. The apparent molecular weight is shown on the left. (D) Western blot analysis in the presence of WIC79.3 antibody to detect LPG. Equivalent of 106 cells were loaded in each lane. (B, C, D): 1, Δlmdat/Δlmdat; 2, Δlmdat/Δlmdat [HV-LmDAT NEO]; 3, Δlmdat/Δlmdat [HV-ΔN546-LmDAT NEO]; 4, Δlmdat/Δlmdat [HV-ΔN686-LmDAT NEO]; 5, Δlmdat/Δlmdat [HV-LmDAT-ΔC733 NEO]; 6, Δlmdat/Δlmdat [HV-LmDAT-ΔC3 NEO]; 7, Δlmdat/Δlmdat [HV-LmDATK852L NEO]; WT, wild type.
Figure 3.
Cells were inoculated at a cell density of 5×105/ml and were enumerated with a hemacytometer as a function of time. The assay was performed twice and the graphs represent a typical experiment. Standard deviations are shown. (A) Black circles, wild type; grey circles, complemented line Δlmdat/Δlmdat [HV-LmDAT NEO]; white circles, Δlmdat/Δlmdat; white triangles, Δlmdat/Δlmdat [HV-ΔN546-LmDAT NEO]; grey triangles, Δlmdat/Δlmdat [HV-ΔN686-LmDAT NEO]; black triangles, Δlmdat/Δlmdat [HV-LmDAT-ΔC733 NEO]. (B) Black circles, wild type; grey circles, complemented line Δlmdat/Δlmdat [HV-LmDAT NEO]; white circles, Δlmdat/Δlmdat; white triangles, Δlmdat/Δlmdat [HV-LmDAT-ΔC3 NEO]; grey triangles, Δlmdat/Δlmdat [HV-LmDATK852L NEO].
Figure 4.
HV-LmDAT-ΔC3 does not localize in the glycosomes.
Wild type expressing recombinant HV-LmDAT-ΔC3 was analyzed by phase contrast (panel 1) or immunofluorescence microscopy using anti-V5 antibody (panel 2) or polyclonal antiserum specific to hypoxanthine guanine phosphoribosyltransferase (panel 3). Panel 4 shows the merge of panels 2 and 3.
Figure 5.
Digitonin fractionation followed by Western blot analysis.
FV1 [HV-LmDAT NEO] and FV1 [HV-LmDAT-ΔC3 NEO] were fractionated in the presence of digitonin as described in Materials and Methods. Cell supernatants were then subjected to Western blot analysis in the presence of monoclonal anti-V5 antibodies (V5), and of polyclonal immunoglobulins specific to arginase (ARG) and phosphomannomutase (PMM). Equivalent of 107 cell supernants were loaded in each lane. The apparent molecular weight markers are shown.