title: Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization.
abstract: BACKGROUND
  Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated.
  
  
  METHODS
  The presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR). Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp.
  
  
  RESULTS
  Semi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed) is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP) co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt.
  
  
  CONCLUSIONS
  Distinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Wichai
authors.initials: W
authors.lastname: Pornthanakasem
authors.forename: Darin
authors.initials: D
authors.lastname: Kongkasuriyachai
authors.forename: Chairat
authors.initials: C
authors.lastname: Uthaipibull
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.forename: Ubolsree
authors.initials: U
authors.lastname: Leartsakulpanich
mesh_terms: D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D020043:Genes, Essential; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling
chemical_list: D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D012696:Glycine Hydroxymethyltransferase