Abstract
Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P . yoelii . The composition of food vacuolar proteome and likely interactors of enolase are also being reported.
Highlights
Enolase is one of the most abundant glycolytic enzymes in the cytosol
Analysis of organellar proteomes by mass spectrometry tends to identify a large number of minor contaminants that are usually not observed in western analysis [13,35,36]
Both these lanes showed reactivity with a protein band about 90 kDa. This band was not observed in the antibody pull down assays [Figure 1C (b), (c) & (d)] and is likely to be a non-specific interaction. These results suggest that the two high molecular mass enolase positive bands (i.e. 65 and 75 kDa) associated with food vacuole (FV) might arise due to conjugation of enolase with ubiquitin
Summary
Enolase is one of the most abundant glycolytic enzymes in the cytosol. It catalyzes the inter-conversion of 2phosphoglycerate and phosphoenolpyruvate during glycolysis and gluconeogenesis, respectively. Apart from its cytosolic localization, the presence of this protein has been reported on the plasma membrane [1,2,3,4,5,6], nucleus [7,8,9,10,11] and vacuole [5,12,13] This diverse sub-cellular distribution of enolase is likely to be associated with a variety of different functions, e.g. serving as a plasminogen receptor on the cell surface [2,14], ligand for mosquito gut epithelial receptors [15,16], transcription factor in plants [8] and cancer cells [9], a component of RNA degradosome in E. coli [17], inhibitor of Dnmt in Entamoeba histolytica [7], structural component of eye lens [18], heat shock protein in yeast [19] etc. Adaptive changes in the proteome of P. falciparum merozoites on switching the invasion dependence from sialated to nonsialated receptor on erythrocytes, showed up-regulation of enolase [22] Another invasive stage of the parasite, ookinete that invades the mosquito gut wall has cell surface localized enolase. Both these functional roles are important for the invasion of the mosquito gut wall by ookinete
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