Acylation-dependent Export of Trypanosoma cruzi Phosphoinositide-specific Phospholipase C to the Outer Surface of Amastigotes

Abstract

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.

Ca 2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C

“…Previous studies with TcPI-PLC (8) revealed that the N-terminal 20 amino acids of the protein have the necessary and sufficient information to target GFP to the surface of cells. We therefore investigated whether this was also the case with TbPI-PLC1.…”

“…1B). The first 18 amino acids of the TbPI-PLC1 sequence, TbPI-PLC1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) , fused to GFP, and the truncated PI-PLC1 protein, TbPI-PLC1 (19 -714) , were transfected into PCF (strain 29-13) trypanosomes. Cells were induced by the addition of tetracycline (1 g/ml), and lysates were analyzed by Western blotting for expression of a protein of the expected size with anti-GFP (Fig.…”

“…For example, they have a myristoylation consensus sequence at the N terminus, instead of a pleckstrin homology (PH) domain (7), and this sequence has been shown to play a role in the localization of the enzyme to the plasma membrane of T. cruzi (8). A very interesting characteristic of the trypanosomatid enzymes is that their overall domain organization is most similar to that of the mammalian zeta-type PI-PLC (PI-PLC-) present in sperm (9).…”

“…Parasites were blocked for 1 h at room temperature with a blocking solution containing 50 mM NH 4 Cl, 3% bovine serum albumin (BSA), 1% fish gelatin, and 5% goat serum. Cells were incubated with a Roche monoclonal anti-HA antibody (1:20) and either the polyclonal rabbit antibody against TbVP1 (1:300), the rabbit anti-TbBiP antibody (1:300), the mouse anti-TbPPDK antibody (1:30), or the mouse monoclonal anti-PIP 2 antibody (1:50) for 1 h. The secondary antibodies were Alexa 488-conjugated goat anti-rat (anti-HA) and/or Alexa 546-conjugated goat anti-rabbit/mouse at 1:1,000 for 1 h. All images of parasites (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) ::GFP (NtGFP), and C-terminal TbPI-PLC1 (19 -714) ::GFP (CtGFP) expressed in PCF trypanosomes. Lysates containing 30 g protein were subjected to SDS-PAGE on 10% polyacrylamide gels and transferred to a nitrocellulose membrane.…”

“…The inserted fragment of VSG117 cDNA was removed by digestion with HindIII and BamHI. First, a primer pair to amplify the coding region for the first 18 amino acids, i.e., the TbPI-PLC1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) primer pair (Table 1), was generated to incorporate HindIII and XhoI sites. Second, a pair of primers for the GFP tag was used to amplify the open reading frame (ORF) of Aequorea victoria GFP, incorporating an XhoI site and a BamHI site ( Table 1).…”