Abstract
N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) CAB10575
It has been shown that the transport of some proteins from malaria parasites is sensitive to BFA [12,13,14,15,16], which is a well known macrolide antibiotic produced by fungi that blocks eukaryotic protein trafficking processes, especially transport from the endoplasmic reticulum to the Golgi apparatus by inhibiting the activities of ADP-ribosylation factors and guanine nucleotide exchange factors [17]
Part of P. falciparum N-Ethylmaleimide-sensitive factor (NSF) (PfNSF) appears to be exported from the parasite cells and localized in vesicular structures in the erythrocyte cytoplasm in a BFA-sensitive manner. These results are consistent with the idea that a supramolecular protein complex consisting of NSF, soluble NSF-attachment protein (SNAP), and receptors for soluble NSF attachment protein is involved in the targeting of P. falciparum proteins to the plasma membranes of host erythrocytes
Summary
PfEMP, erythrocyte membrane protein of Plasmodium falciparum; BFA, brefeldin A; C5-ceramide, N-(4,4difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine; NSF, N-ethylmaleimide-sensitive factor; PfNSF, N-ethylmaleimide-sensitive factor from P. falciparum; PBS, phosphatebuffered saline; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; CHO, Chinese hamster ovary; MOPS, 4-morpholinepropanesulfonic acid; RT-PCR, reverse transcription-polymerase chain reaction Part of P. falciparum NSF (PfNSF) appears to be exported from the parasite cells and localized in vesicular structures in the erythrocyte cytoplasm in a BFA-sensitive manner These results are consistent with the idea that a supramolecular protein complex consisting of NSF, SNAP, and receptors for soluble NSF attachment protein is involved in the targeting of P. falciparum proteins to the plasma membranes of host erythrocytes
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