TY - JOUR
T1 - The primase domain of PfPrex is a proteolytically matured, essential enzyme of the apicoplast
AU - Lindner, Scott E.
AU - Llinás, Manuel
AU - Keck, James L.
AU - Kappe, Stefan H.I.
N1 - Funding Information:
We thank G. McFadden for providing the anti-PfACP antibody, and J. Berger for purified recombinant EcDnaG. We also thank the Keck and Kappe labs for critical discussion of this work. Special thanks to Drew MacKellar for microscopy assistance, and to Nelly Camargo for training in the transfection of P. falciparum . This investigation was supported by the NIH under a Ruth L. Kirschstein National Research Service Award ( F32GM083438 ) to SEL and by an R01 award ( GM068061 ) to JLK. SEL performed research and wrote the report; SEL, JLK, ML, and SK designed research, and analyzed and interpreted data.
PY - 2011/12
Y1 - 2011/12
N2 - The apicoplast of Plasmodium is an essential organelle with its own circular genome that must be faithfully replicated and segregated to its progeny during parasite sporogony and schizogony. DNA replication proteins are not encoded by its genome. Instead, the replication machinery must be imported from nuclear-encoded genes. A likely apicoplast DNA replication factor, PfPrex, bears a bipartite leader sequence for apicoplast trafficking and contains several DNA replication-related enzymatic domains. Here we analyze the domain structure of PfPrex and examine its trafficking and maturation within the parasite. A minimal primase domain of PfPrex is shown to contain functional zinc-binding and TOPRIM-fold domains, which in a recombinant form are sufficient to produce RNA primers from a single-stranded DNA template. PfPrex is shown to be extensively proteolytically matured within the parasite, which effectively separates its functional domains. Gene targeting attempts to knockout the Plasmodium yoelii ortholog of Prex were unsuccessful, indicating the apparent essentiality of this protein to the parasite. Finally, overexpression in Plasmodium falciparum of PfPrex's trafficking and primase sequences yielded specific and dynamic localization to foci within the apicoplast. Taken together, these observations strongly suggest an essential role of PfPrex primase in the production of RNA primers for lagging strand DNA synthesis of the apicoplast genome.
AB - The apicoplast of Plasmodium is an essential organelle with its own circular genome that must be faithfully replicated and segregated to its progeny during parasite sporogony and schizogony. DNA replication proteins are not encoded by its genome. Instead, the replication machinery must be imported from nuclear-encoded genes. A likely apicoplast DNA replication factor, PfPrex, bears a bipartite leader sequence for apicoplast trafficking and contains several DNA replication-related enzymatic domains. Here we analyze the domain structure of PfPrex and examine its trafficking and maturation within the parasite. A minimal primase domain of PfPrex is shown to contain functional zinc-binding and TOPRIM-fold domains, which in a recombinant form are sufficient to produce RNA primers from a single-stranded DNA template. PfPrex is shown to be extensively proteolytically matured within the parasite, which effectively separates its functional domains. Gene targeting attempts to knockout the Plasmodium yoelii ortholog of Prex were unsuccessful, indicating the apparent essentiality of this protein to the parasite. Finally, overexpression in Plasmodium falciparum of PfPrex's trafficking and primase sequences yielded specific and dynamic localization to foci within the apicoplast. Taken together, these observations strongly suggest an essential role of PfPrex primase in the production of RNA primers for lagging strand DNA synthesis of the apicoplast genome.
UR - http://www.scopus.com/inward/record.url?scp=80054700244&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80054700244&partnerID=8YFLogxK
U2 - 10.1016/j.molbiopara.2011.08.002
DO - 10.1016/j.molbiopara.2011.08.002
M3 - Article
C2 - 21856338
AN - SCOPUS:80054700244
SN - 0166-6851
VL - 180
SP - 69
EP - 75
JO - Molecular and biochemical parasitology
JF - Molecular and biochemical parasitology
IS - 2
ER -