Food vacuole associated enolase in plasmodium undergoes multiple post-translational modifications: evidence for atypical ubiquitination.

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

Plasmodium falciparum enolase complements yeast enolase functions and associates with the parasite food vacuole

Plasmodium falciparum enolase (Pfeno) localizes to the cytosol, nucleus, cell membrane and cytoskeletal elements, suggesting multiple non-glycolytic functions for this protein. Our recent observation of association of enolase with the food vacuole (FV) in immuno-gold electron microscopic images of P. falciparum raised the possibility for yet another moonlighting function for this protein. Here we provide additional support for this localization by demonstrating the presence of Pfeno in purified FVs by immunoblotting. To examine the potential functional role of FV-associated Pfeno, we assessed the ability of Pfeno to complement a mutant Saccharomyces cervisiae strain deficient in enolase activity. In this strain (Tetr-Eno2), the enolase 1 gene is deleted and expression of the enolase 2 gene is under the control of a tetracycline repressible promoter. Enolase deficiency in this strain was previously shown to cause growth retardation, vacuolar fragmentation and altered expression of certain vacuolar proteins. Expression of Pfeno in the enolase-deficient yeast strain restored all three phenotypic effects. However, transformation of Tetr-eno2 with an enzymatically active, monomeric mutant form of Pfeno (Δ5Pfeno) fully restored cell growth, but only partially rescued the fragmented vacuolar phenotype, suggesting that the dimeric structure of Pfeno is required for the optimal vacuolar functions. Bioinformatic searches revealed the presence of Plasmodium orthologs of several yeast vacuolar proteins that are predicted to form complexes with Pfeno. Together, these observations raise the possibility that association of Pfeno with food vacuole in Plasmodium may have physiological function(s)

Ubiquitin moiety in <i>P</i><i>. yoelii</i>.

<p>(A) <i>P</i><i>yoelii</i> hypothetical protein (NCBI id: gi| 82539669; or Plasmodb id: PY03971 hypothetical protein) has four ubiquitin like internal repeats. Residues in blue, red, green and magenta belongs to peptides that were identified in MS analysis of tryptic digest of ~65-80 kDa band from FV gel. (B) Schematic representation of four ubiquitin repeats with cleavage sites shown in red arrows. Note that first subunit has an insert of 16 residues (aa11-26) raising its MW to ~92 kDa. Last repeat C-terminus has –GV instead of a –GG. (C) MS/MS spectrum of a peptide fromputative ubiquitin of <i>P</i><i>. yoelii</i> putative ubiquitin. This peptide was present in the tryptic digest of the food vacuole SDS-PAGE 65-80 kDa band (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072687#pone-0072687-g001" target="_blank">Figure 1</a>). The peptide showed a linear peptide linkage between –G76 and M1 of two µb (Ub1 and Ub2) molecules while the K6 of Ub2 is the site for attachment of third Ub (Ub3) molecule. (D) CLUSTAL W alignment of four repeats from PY03971 protein and human ubiquitin. Repeats 2 and 3 have the same sequence as that of human (and mouse) except E16 in human (and mouse) is replaced with a D in <i>Plasmodium</i><i>spp</i>. Observation of ‘D16’ in sequenced peptides [see (A)] indicates the Ub linked to protein in ~65-80 kDa band is of parasite origin and not that of host.</p

Analysis of <i>P</i><i>. yoelii</i> food vacuole (FV) associated enolase variants using antibody pull down assays and western blots.

<div><p>(A) <i>P</i><i>. yoelii</i> cell fractionation scheme. Various fractions used for analysis are marked in bold. Proteins were separated on 10% SDS PAGE.</p> <p>(B) Western blot of FV proteome probed with (a) anti-rPfeno (mouse) and anti-Ub (mouse) antibodies. Note that three variants of enolase were observed while two of these (65 & 75 kDa) are ubiquitinated (dotted line box). Matched amounts of proteins were loaded in both the lanes.</p> <p>(C) Antibody pull-down assays for different sub-cellular fractions using anti-rPfeno (mouse) and anti-Ub antibodies (mouse). Blots of pull-down proteins were probed with anti-rPfeno (rabbit) to detect the presence of enolase.</p> <p>(D) Western blots of whole cell soluble fraction and total FV probed with ant-PfP0 (cytosolic marker) and anti-falcipain-2 (FV marker) antibodies. Equivalent amount of total proteins were loaded in each lane. Note the enrichment of falcipain-2 and near absence of P0 in FV preparation indicating that preparation used in above experiments is highly enriched in FV.</p></div

MS/MS spectrum of a peptide from <i>P</i><i>. yoelii</i> enolase (Pyeno).

<p>Sequence covers residues 136-155 of Pyeno. Three different post-translational modifications are observed. K142 is acetylated, K147 has attached glycylglycyl residues through a trans-peptide linkage (a signature of ubiquitination) and Y148 is phosphorylated. ‘b’ ions corresponding to relevant modifications are marked with arrows along with aminoacyl residue mass + Δm for the modifying group.</p