Phosphoinositide 5-Phosphatase Fig4p Is Required for both Acute Rise and Subsequent Fall in Stress-Induced Phosphatidylinositol 3,5-Bisphosphate Levels

Abstract

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P 2 ) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P 2 , vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P 2 vacuole volume is restored. We show that Fig4p, consistent with its proposed role as a PI3,5P 2 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P 2 . Surprisingly, we find that Fig4p is also required for the hyperosmotic shock-induced increase in PI3,5P 2 levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P 2 and further suggest that Fig4p is important in regulating both the acute rise and subsequent fall in PI3,5P 2 levels.

Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

“…This is also suggested by studies in yeast, which demonstrated an in vitro or in vivo specificity of the Fig4 phosphatase for position 5 in PtdIns(3,5)P 2 substrate and a marked up-regulation of PtdIns(3,5)P 2 upon expression of Fig4 mutants with substitutions within the Sac phosphatase domain (11,18,41,58). To examine whether Sac3, like Fig4, hydrolyzes PtdIns(3,5)P 2 , we conducted experiments both in vitro and in a cell context.…”

“…The consensus CX 5 R(T/S) active site, invariant in mammalian Sac domain containing phosphatases, is positioned within the sixth motif with a sequence 486 CVDCLDRTN. Accordingly, the Sac3 D488A mutant, similarly to a corresponding mutation in Fig4 (11,41), is devoid of phosphatase activity as measured in vitro by the malachite green assay (see below). Unlike the Sac1 proteins, yeast Fig4 and mammalian Sac3 do not display a putative transmembrane sequence, and besides the regions of the Sac phosphatase domain and low complexity, there are no other obvious conserved domains along the Fig4/ Sac3 molecule ( Fig.…”

“…It should be emphasized, however, that regulation of PtdIns(3,5)P 2 levels could occur by both synthesis and turnover. Concordantly, a Sac domain-containing 5-phosphatase, Fig4, has been recently characterized in budding yeast and found to turn over PtdIns(3,5)P 2 to PtdIns(3)P both in vitro and in vivo (11,18,41). Reportedly, Fig4 directly interacts with Vac14, which promotes its localization to the site of PtdIns(3,5)P 2 synthesis (11,40).…”

Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice

“…PI(3,5)P 2 is synthesized from PI(3)P by the PI(3)P 5-kinase Fab1/PIKfyve/PIP5K3 (5,6). Fab1 is stimulated by a regulatory complex that contains Vac14 (7,8) and Fig4 (4,9). Surprisingly, the Vac14/Fig4 complex plays two opposing roles in the regulation of steady-state levels of PI(3,5)P 2 .…”

rSac3, a new member of Sac domain phosphoinositide phosphatases family

“…The major cellular function of Sac domain-containing proteins is to modulate intracellular membrane trafficking, including exocytosis, endocytosis and membrane transportation between various intracellular compartments, via hydrolyzing their corresponding phosphoinositide substrates [1,2,7,8]. Synaptojanin 1 and 2 are involved in regulating endocytic vesicle fission and phosphoinositide dephosphorylation [1].…”