A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.
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.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
M
M
Hayashi
S
S
Taniguchi
Y
Y
Ishizuka
H S
HS
Kim
Y
Y
Wataya
A
A
Yamamoto
Y
Y
Moriyama
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A / Q000494:pharmacology*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D004912:Erythrocytes / Q000378:metabolism* / Q000469:parasitology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000378:metabolism; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D020126:Brefeldin A