Browsing by Author "Speicher, Kaye D."

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    Endoplasmic reticulum PI(3)P lipid binding targets malaria proteins to the host cell
    (Elsevier, 2012) Bhattacharjee, Souvik; Stahelin, Robert V.; Speicher, Kaye D.; Speicher, David W.; Haldar, Kasturi; Biochemistry and Molecular Biology, School of Medicine
    Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a "secretome" carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here, we show that the HT signal facilitates export by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants, bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER and colocalize with endogenous HT signal on ER precursors, which also display high-affinity binding to PI(3)P. A related pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus, PI(3)P in the ER functions in mechanisms of secretion and pathogenesis.
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    PI(3)P-independent and -dependent pathways function together in a vacuolar translocation sequence to target malarial proteins to the host erythrocyte
    (Elsevier, 2012) Bhattacharjee, Souvik; Speicher, Kaye D.; Stahelin, Robert V.; Speicher, David W.; Haldar, Kasturi; Biochemistry and Molecular Biology, School of Medicine
    Malaria parasites export ‘a secretome’ of hundreds of proteins, including major virulence determinants, from their endoplasmic reticulum (ER), past the parasite plasma and vacuolar membranes to the host erythrocyte. The export mechanism is high affinity (nanomolar) binding of a host (cell) targeting (HT) motif RxLxE/D/Q to the lipid phosphatidylinositol 3-phosphate (PI(3)P) in the ER. Cleavage of the HT motif releases the secretory protein from the ER membrane. The HT motif is thought to be the only export signal resident in an N-terminal vacuolar translocation sequence (VTS) that quantitatively targets green fluorescent protein to the erythrocyte. We have previously shown that the R to A mutation in the HT motif, abrogates VTS binding to PI(3)P (Kd > 5 μM). We now show that remarkably, the R to A mutant is exported to the host erythrocyte, for both membrane and soluble reporters, although the efficiency of export is reduced to ~ 30% of that seen with a complete VTS. Mass spectrometry indicates that the R to A mutant is cleaved at sites upstream of the HT motif. Antibodies to upstream sequences confirm that aberrantly cleaved R to A protein mutant is exported to the erythrocyte. These data suggest that export mechanisms, independent of PI(3)P as well as those dependent on PI(3)P, function together in a VTS to target parasite proteins to the host erythrocyte.