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.


Available from: Yeun Ju Kim, Jul 09, 2014

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

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    • "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 "
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    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.
    Developmental Cell 05/2015; 33(5). DOI:10.1016/j.devcel.2015.04.028 · 9.71 Impact Factor
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    • "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 . "
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    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.
    The Journal of General Physiology 02/2014; 143(2):183-201. DOI:10.1085/jgp.201311033 · 4.79 Impact Factor
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    • "The ER is a complex system of membranes consisting of tubules and cisternae, and is constantly extending and making contact with other organelles. A recent study has suggested that PIS is found in a mobile sub-compartment of the ER that may be the site of PI synthesis (Kim et al., 2011). Formation of this domain is dependent on Sar1 activity. "
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    ABSTRACT: The hallmark of mammalian phosphatidylinositol transfer proteins (PITPs) is to transfer phosphatidylinositol between membrane compartments. In the mammalian genome, there are three genes that code for soluble PITP proteins, PITPα, PITPβ and RdgBβ and two genes that code for membrane-associated multi-domain proteins (RdgBαI and II) containing a PITP domain. PITPα and PITPβ constitute Class I PITPs whilst the RdgB proteins constitute Class II proteins based on sequence analysis. The PITP domain of both Class I and II can sequester one molecule of phosphatidylinositol (PI) in its hydrophobic cavity. Therefore, in principle, PITPs are therefore ideally poised to couple phosphatidylinositol delivery to the PI kinases for substrate provision for phospholipases C during cell activation. Since phosphatidylinositol (4,5)bisphosphate plays critical roles in cells, particularly at the plasma membrane, where it is a substrate for both phospholipase C and phosphoinositide-3-kinases as well as required as an intact lipid to regulate ion channels and the actin cytoskeleton, homeostatic mechanisms to maintain phosphatidylinositol(4,5)bisphosphate levels are vital. To maintain phosphatidylinositol levels, phospholipase C activation inevitably leads to the resynthesis of PI at the endoplasmic reticulum where the enzymes are located. Phosphatidic acid generated at the plasma membrane during phospholipase C activation needs to move to the ER for conversion to PI and here we provide evidence that Class II PITPs are also able to bind and transport phosphatidic acid. Thus RdgB proteins could couple PA and PI transport bidirectionally during phospholipase C signalling.
    07/2013; 53(3). DOI:10.1016/j.jbior.2013.07.007
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