A Highly Dynamic ER-Derived Phosphatidylinositol-Synthesizing Organelle Supplies Phosphoinositides to Cellular Membranes

Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, MD 20892, USA.
Developmental Cell (Impact Factor: 9.71). 11/2011; 21(5):813-24. DOI: 10.1016/j.devcel.2011.09.005
Source: PubMed


Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.

  • Source
    • "Rather, the rate-limiting enzyme is phosphatidylinositol synthase (PIS), which is encoded by a single ubiquitously expressed gene[207]. GFP-tagged PIS localizes to the tubular ER network with CDS but is also present in an unusual, high-mobility membrane/vesicular compartment that emanates from the ER by a Sar1-dependent mechanism and fleetingly contacts PM-ER junctions that mediate store operated calcium entry[211]. Based on density gradient fractionation, the mobile vesicle fraction has greater PIS activity compared to the bulk ER and only accommodates catalytically active enzyme. "

    Full-text · Chapter · Jan 2016
  • Source
    • "This was tested by using DiC 8 -DG added to the cells expressing the GFP-Nir2(420– 1181) or GFP-Nir2(816–1181) constructs. This treatment caused rapid translocation of both the DG sensor (Kim et al., 2011) and the Nir2 construct (Figure 6E) to the membrane, even after "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sustained agonist-induced production of the second messengers InsP3 and diacylglycerol requires steady delivery of phosphatidylinositol (PtdIns) from its site of synthesis in the ER to the plasma membrane (PM) to maintain PtdIns(4,5)P2 levels. Similarly, phosphatidic acid (PtdOH), generated from diacylglycerol in the PM, has to reach the ER for PtdIns resynthesis. Here, we show that the Drosophila RdgB homolog, Nir2, a presumed PtdIns transfer protein, not only transfers PtdIns from the ER to the PM but also transfers PtdOH to the opposite direction at ER-PM contact sites. PtdOH delivery to the ER is impaired in Nir2-depleted cells, leading to limited PtdIns synthesis and ultimately to loss of signaling from phospholipase C-coupled receptors. These studies reveal a unique feature of Nir2, namely its ability to serve as a highly localized lipid exchanger that ensures that PtdIns synthesis is matched with PtdIns(4,5)P2 utilization so that cells maintain their signaling competence. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · May 2015 · Developmental Cell
  • Source
    • "How­ ever, in case of receptor­operated TRPC6/7 currents, we observed that a high concentration of ATP or its in­ active analogue AMP­PNP in the patch­pipette solution had little effect on TRPC6/7 currents (Fig. S7), even in the plateau or biphasic phase. We speculate that an ad­ ditional PI(4,5)P 2 replenishment pathway, such as the detachment of PI(4,5)P 2 from proteins, dispersion from its clustered complex (van den Bogaart et al., 2011), translocation of phosphoinositides from the PIS organelle (Kim et al., 2011), or an unknown mechanism (Hammond et al., 2012), may be involved. However, here we focused on the lateral diffusion of PI(4,5)P 2 . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.
    Full-text · Article · Feb 2014 · The Journal of General Physiology
Show more