A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization.
Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.
Chan
Maurice
M
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597, Singapore.
Tan
Doreen S H
DS
Wong
Siew-Heng
SH
Sim
Tiow-Suan
TS
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002950:Citrate (si)-Synthase / Q000378:metabolism; D002952:Citric Acid Cycle; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008928:Mitochondria / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010963:Plasmodium falciparum / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D015800:Protozoan Proteins / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism
D010455:Peptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007521:Isocitrate Dehydrogenase; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D013385:Succinate Dehydrogenase; D002950:Citrate (si)-Synthase; D006655:Histone Deacetylases