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Plasmodium spp. Glyceraldehyde 3-phosphate dehydrogenase: Diverse subcellular localization and multiple post-translational modifications

ABSTRACT: Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), a glycolytic enzyme is known to be associated with multiple non-glycolytic functions. To examine the possibility of Plasmodium spp Gapdh having moonlighting functions, PfGapdh was cloned, overexpressed, purified to homogeneity and characterized. Polyclonal antibodies were raised in mice and used for immuno detection. Western analysis of P. yoelii whole cell extract, soluble and particulate fractions showed three major species with molecular mass ~27, 37 and 51 kDa. All three forms were equally abundant in the soluble fraction. Particulate fraction that accounted for ~10-15% of total PyGapdh did not show ~51 kDa form. Immunological evidence suggests~51 kDa form to arise by ubiquitination of native 37kDa form. For determining subcellular distribution, P. yoelii cells were subjected to differential detergent fractionation and analyzed using two-dimensional gel electrophoresis. Various subcellular fractions showed multiple species of Gapdh, indicating multiple post-translational modifications. For the determination of the nature of PTMs and residues modified, ‘Top-Down’ and ‘Bottom-Up’ approaches were used. Spots in a 2D gel that showed high intensity in western were excised and analyzed for identification of protein as well as PTMs. For more comprehensive analysis of PTM, P. yoelii whole cell extract was run on a 1D gel and bands that showed reactivity in western were cut out and analyzed by LC-MS/MS. In all, eleven residues were identified that underwent modifications. Modifications consisted of four phosphorylations, five ubiquitinations, two acetylations, two methylations and one nitrosylation. Physiological relevance of these modifications is not yet understood

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Plasmodium Yoelii Yoelii

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Nitin Jindal

LAB HEAD: Gotam K Jarori

PROVIDER: PXD002313 | Pride | 2018-01-08

REPOSITORIES: Pride

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Project description:Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems toward understanding the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be differentially expressed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as isoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of small G protein Ran1, 14-3-3 protein, actin, and Caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings provide up-to-date evidences for understanding fern spore asymmetric division and rhizoid polar growth.

			Action	DRS
	1.d.zip	Other
	1.dat	Other
	1.dat-pride.pride.mgf.gz	Mgf
	1.dat-pride.pride.mztab.gz	Mztab
	1.dat-pride.xml.gz	Xml

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Plasmodium spp. Glyceraldehyde 3-phosphate dehydrogenase: Diverse subcellular localization and multiple post-translational modifications

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