Plentiful PtdIns5P from scanty PtdIns(3,5)P₂ or from ample PtdIns? PIKfyve‐dependent models: Evidence and speculation (response to: DOI 10.1002/bies.201300012)

Abstract

Recently we have presented data supporting the notion that PIKfyve not only produces the majority of constitutive phosphatidylinositiol 5-phosphate (PtdIns5P) in mammalian cells but that it does so through direct synthesis from PtdIns. Another group, albeit obtaining similar data, suggests an alternative pathway whereby the low-abundance PtdIns(3,5)P2 undergoes hydrolysis by unidentified 3-phosphatases, thereby serving as a precursor for most of PtdIns5P. Here, we review the experimental evidence supporting constitutive synthesis of PtdIns5P from PtdIns by PIKfyve. We further emphasize that the experiments presented in support of the alternative pathway are also compatible with a direct mechanism for PIKfyve-catalyzed synthesis of PtdIns5P. While agreeing with the authors that constitutive PtdIns5P could theoretically be produced from PtdIns(3,5)P2 by 3-dephosphorylation, we argue that until direct evidence for such an alternative pathway is obtained, we should adhere to the existing experimental evidence and quantitative considerations, which favor direct PIKfyve-catalyzed synthesis for most constitutive PtdIns5P.

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis

“…The ramification of the lost PIKfyve endosome localization is a commensurate reduction of PtdIns5P production (Fig. 4), a finding consistent with direct PIKfyve-catalyzed synthesis of PtdIns5P (46,54). The concomitant PtdIns5P diminution suggests that the cytosolic PIKfyve fails to gain access to the PtdIns substrate on membranes and/or form a productive heterooligomeric complex (PAS complex) with the two partner proteins, i.e., the scaffolding regulator ArPIKfyve and the antagonistic PtdIns(3,5)P 2 phosphatase Sac3 (1,37,55).…”

“…Both membrane recruitment and the PAS complex formation are crucial steps for PIKfyve activity, as evidenced by the inability of the dominant-negative kinase-deficient point mutant PIKfyve K1831 to trigger the vacuolation phenotype if incapacitated for PtdIns3P binding or ArPIKfyve-Sac3 association through truncated mutations in the FYVE domain or ArPIKfyve-Sac3 interaction sites, respectively (9,56). Concordantly, gene disruption of ArPIKfyve or Sac3 impairs PtdIns5P synthesis, further reinforcing the prerequisite of intact PAS complex in PIKfyve-catalyzed PtdIns5P production [reviewed in (46,54)].…”

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

“…21 PIKfyve is an Fyve (Fab1p, YOTB, Vac1p, and early endosome antigen 1 [EEA1]) finger-containing phosphoinositide (PI) kinase that phosphorylates PtdIns and Vps34-generated PtdIns(3)P on the 5-hyrdroxyl position to make the low-abundance signaling lipids phosphatidylinositol-5-phosphate [PtdIns(5)P]and phosphatidylinositol-3,5-phosphate 2 [PtdIns(3,5)P2], respectively. [22][23][24] In contrast to PtdIns(3)P, which is found in early endosomal compartments, PtdIns (3,5)P2 is thought to predominate in late endosomes and/or lysosomes. [25][26][27][28] Similar to Vps34, perturbations in PIKfyve-catalyzed PtdIns (3,5)P2 synthesis lead to several defects, including a dramatic enlargement of endosomal/lysosomal membranes.…”

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

“…The slope of PI(4,5)P 2 formation from PI5P was 20 times steeper than that of PI(3,4)P 2 formation from PI3P over a nanomole concentration range of each substrate ( Figure 2B). Lipid samples prepared from cells for the kinase assay will have a mixture of PI5P and PI3P, wherein the levels of PI3P would be equal or higher than that of PI5P by approximately two to three times [13,27].…”