Sub-cellular localization and post-translational modifications of the Plasmodium yoelii enolase suggest moonlighting functions.
BACKGROUND
Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii.


METHODS
Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts.


RESULTS
Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelii. Different sub-cellular fractions showed different isoform profiles.


CONCLUSIONS
Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions.
Pal-Bhowmick
Ipsita
I
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India. ipsita@tifr.res.in <ipsita@tifr.res.in>
Vora
Hardeep K
HK
Jarori
Gotam K
GK
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D003599:Cytoskeleton / Q000201:enzymology; D005455:Fluorescent Antibody Technique / Q000379:methods; D007527:Isoenzymes / Q000378:metabolism; D008288:Malaria / Q000469:parasitology; D051379:Mice; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D010766:Phosphorylation; D010967:Plasmodium yoelii / Q000201:enzymology* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D010751:Phosphopyruvate Hydratase