A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Abstract

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H ؉ -ATPase and vacuolar H ؉ -pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.Phosphatidylinositol 3-kinase (PI3K) 6 activities have been found in all eukaryotic cell types examined to date (1, 2) and are linked to a diverse set of key cellular functions, including cell growth, survival, and intracellular trafficking. PI3Ks belong to a large family of enzymes that has been divided into three functional classes on the basis of their protein domain structure, lipid substrate specificity, and regulatory properties. Class I PI3Ks were the first ones to be identified and are important components of the signaling pathways that regulate eukaryotic cell growth (3, 4). These PI3Ks have a 110-kDa catalytic subunit that exhibits a substrate preference for PI 4-phosphate and PI 4,5-bisphosphate (5). Class II PI3Ks are less well known but may also function in the regulation of cell growth (5, 6) and, additionally, in clathrin-mediated endocytosis (7, 8). These enzymes prefer phosphatidylinositol (PI) as substrate but may also utilize PI 4-phosphate. Finally, class III family of PI3Ks is related to the yeast vacuolar protein sorting 34, Vps34p, and their homologs from other eukaryotes. Vps34p-like kinases specifically phosphorylate PI to produce phosphatidylinosi...

Delayed Phosphorylation of Classical Protein Kinase C (PKC) Substrates Requires PKC Internalization and Formation of the Pericentrion in a Phospholipase D (PLD)-dependent Manner

“…The H ϩ -ATPase has been also found to reside in the endosomes. The activities of vacuolar-H ϩ -ATPase and vacuolar H ϩ -pyrophosphatase are altered by GPCRs and tyrosine kinase receptors, confirming a synergetic role in membrane trafficking and in clathrin-mediated endocytosis (37). Their role in the endosomes is to facilitate the dissociation of ligand from receptors by maintaining a low pH in the endosomes (38).…”

Expansion of the target of rapamycin (TOR) kinase family and function in Leishmania shows that TOR3 is required for acidocalcisome biogenesis and animal infectivity

“…Both TOR kinases and ACs have been implicated in sensing environmental signals including nutrients and osmotic stress (2-4, 35). tor3 − was exposed to hypo-osmotic stress, and changes in cell volume were monitored by light scattering, in which a decrease in absorbance corresponds to an increase in volume (36). As expected, in WT and tor3 − /+TOR3 promastigotes, the volume quickly increased, peaking after 1 min and recovering after 3 min (Fig.…”

“…Methodology for assays of AC-related phenotype in the tor3 − mutant, such as EM, immunofluorescence, and DAPI staining are detailed in SI Text. Regulatory volume decrease response was measured by light scatter (36). Briefly, 10 8 late log-phase promastigotes were washed twice in isotonic Iso-Cl buffer (20 mM Hepes, pH 7.4, 11 mM glucose, 1 mM CaCl 2 , 0.8 mM MgSO 4 , 137 mM NaCl, 4 mM KCl, 1.5 mM K 2 HPO 4 , and 8.5 mM Na 2 HPO 4 ), and resuspended in 550 μL Iso-Cl, after which 250-μL aliquots were added to cuvettetes containing an equal volumes of Iso-Cl or water (hypotonic stress).…”

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

“…In addition to the cyclic AMP pathway, other signalling systems have been found to be involved in osmoregulation in T. cruzi. Overexpression of a phosphatidylinositol 3-kinase (PI3K, or Vps34) in T. cruzi resulted in morphological and functional alterations related to vesicular trafficking, and the cells were more resistant to hyposmotic stress (Schoijet et al 2008). Interestingly, these cells had large contractile vacuole bladders (Schoijet et al 2008).…”