Evidence for prenylation-dependent targeting of a Ykt6 SNARE in Plasmodium falciparum

Abstract

Ykt6 proteins are the most versatile fusogens in eukaryotic cells, and the only SNAREs that can be both prenylated and acylated at a C-terminal CAAX motif. Unlike yeast and mammalian cells where a single Ykt6 gene is expressed, the Plasmodium falciparum genome encodes two Ykt6 proteins. We have investigated the expression and prenylation of the Ykt6 orthologue, PfYkt6.1 in intra-erythrocytic stages of P. falciparum. PfYkt6.1 localized to the parasite Golgi and other unidentified cytoplasmic compartments, and was partly cytosolic (∼50% in early trophozoites). The membrane-association of PfYkt6.1 was dependent on the presence of a conserved C-terminal CAAX motif (CCSIM). By expressing full-length and mutant proteins in Escherichia coli, we have shown that PfYkt6.1 indeed serves as substrate for prenylation by P. falciparum farnesyltransferases. Surprisingly, PfYkt6.1 could also be geranylgeranylated by parasite extracts independent of the C-terminal amino acid residue. Deletion of the CAAX motif inhibited both farnesylation and geranylgeranylation activities. Additionally, the PfYkt6.1 heptapeptide KQCCSIM, corresponding to the C-terminal CAAX sequence, inhibited the parasite farnesyltransferase activity with an IC(50) of 1 μM. Our findings underscore the importance of CAAX motif-derived peptidomimetics for antimalarial drug development.

Figure 1. Compartmentalization of PfYkt6.1 in P. falciparum parasites

Trophozoite stage parasites exhibiting increased targeting of the PfYkt6.1 protein to organellar compartments were by confocal immunofluorescence microscopy using various organellar markers. (A) Immunofluorescence micrograph showing complete overlap between the GFP fluorescence and anti-PfYkt6.1 antibody signal in GFP-PfYkt6.1 expressing parasites. The GFP-PfYkt6.1 compartments were only partially labeled with antibodies against the Golgi marker PfErd2 (B), or the ER marker PfBip (C). Scale bars, 2µM.

Figure 2. Detergent solubility of untagged and GFP-tagged PfYkt6.1 proteins

GFP-PfYkt6.1 expressing parasites were solubilized with Triton X-114 and phase separated prior to immunoblot analyses using specific antibodies to the PfYkt6.1 protein (upper left blot) or GFP tag (upper right blot). The lumenal chaperone PfBip and the multi-pass membrane protein PfErd2 were probed as markers for hydrophilic and hydrophobic proteins, respectively. As shown, the untagged and GFP-tagged PfYkt6.1 proteins both partially partitioned into the detergent phase following Triton X-114 phase partitioning. U: untransfected cell lysate, A: Triton X-114 aqueous phase, D: Triton X-114 detergent phase.

Figure 3. Role of the CAAX motif in PfYkt6.1 targeting

(A) Deletion of the C-terminal CAAX sequence CSIM resulted in a diffuse cytosolic localization of the mutant construct, GFP-PfYkt6.1.CSIM. In contrast with the wild-type protein (see Fig. 1), this mutant construct failed to associate with the organellar structures or the ER (B) and Golgi (C) compartments. (D) Triton X-114 phase partitioning of the endogenous PfYkt6.1 protein and GFP-PfYkt6.1.CSIM mutant indicating a complete fractionation of the mutant protein with the Triton X-114 aqueous phase. U: untransfected cell lysate, A: Triton X-114 aqueous phase, D: Triton X-114 detergent phase. Scale bars, 2 µM.

Figure 4. In vitro farnesylation and geranylgeranylation of PfYkt6.1 and CAAX mutants

(A) Dose-dependent inhibition of PfYkt6.1 and mutant construct farnesylation in vitro. (B) Role of the C-terminal residue (position +3 relative to the CAAX cysteine) on the farnesylation efficiency of PfYkt6.1. The +3 residue in each construct is indicated at the top of the respective lanes. (C) Geranylgeranylation efficiency of PfYkt6.1 and mutant constructs. Protein farnesylation was achieved using a Mono Q purified fraction of PfPFTase, whereas geranylgeranylation was obtained using an ammonium sulfate fraction presumably containing the total prenyltransferase activities.

Figure 5. In vitro inhibition of PfPFTase activity with PfYkt6.1 CAAX-containing peptides

Increasing concentrations of the PfYkt6.1 CAAX sequence KQCCSIM, the PfPTP CAAX sequence RKCHFM (potent PfPFTase inhibitor), or the heptapeptide NRSCAIV (negative control) were used to test the farnesylation activity of PfPFTase on the biotinylated lamin B peptide (YRASNRSCAIM). The data represent mean activities ± standard deviations., and are expressed as a percentage of the total enzyme activity in the absence of any competing peptides.