scholarly journals Phosphatidylinositol 4,5-bisphosphate is regenerated by speeding of the PI 4-kinase pathway during long PLC activation

2020 ◽  
Vol 152 (12) ◽  
Author(s):  
Jongyun Myeong ◽  
Lizbeth de la Cruz ◽  
Seung-Ryoung Jung ◽  
Jun-Hee Yeon ◽  
Byung-Chang Suh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Lipid Kinase ◽  
Agonist Action ◽  
Lipid Species ◽  
Phosphoinositide Cycle ◽  
M1 Muscarinic ◽  
Phosphatidylinositol 4 ◽  
Cellular Compartments ◽  
Regulatory Controls

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

scholarly journals A Plasma Membrane Pool of Phosphatidylinositol 4-Phosphate Is Generated by Phosphatidylinositol 4-Kinase Type-III Alpha: Studies with the PH Domains of the Oxysterol Binding Protein and FAPP1

2005 ◽  
Vol 16 (3) ◽  
pp. 1282-1295 ◽  
Author(s):  
Andras Balla ◽  
Galina Tuymetova ◽  
Arnold Tsiomenko ◽  
Péter Várnai ◽  
Tamas Balla
Keyword(s):  
Plasma Membrane ◽  
Down Regulation ◽  
Type Iii ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Cytoplasmic Vesicles ◽  
Ph Domains ◽  
Phosphatidylinositol 4 ◽  
Cellular Compartments ◽  
P Distribution

The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 μM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIα or PI4KIIIβ by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIβ or -IIα, but was inhibited by down-regulation of PI4KIIIα, or by 10 μM PAO, which also inhibits PI4KIIIα. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.

scholarly journals Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3

2008 ◽  
Vol 183 (6) ◽  
pp. 1061-1074 ◽  
Author(s):  
Dan Baird ◽  
Chris Stefan ◽  
Anjon Audhya ◽  
Sabine Weys ◽  
Scott D. Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Essential Gene ◽  
Additional Component ◽  
Lipid Kinase ◽  
Phosphoinositide Kinase ◽  
Multiple Copies ◽  
The Stability ◽  
Phosphatidylinositol 4

The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

scholarly journals Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2

2009 ◽  
Vol 422 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Gerald R. V. Hammond ◽  
Giampietro Schiavo ◽  
Robin F. Irvine
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Plasma Membranes ◽  
Membrane Lipid ◽  
Present Evidence ◽  
Immunocytochemical Detection ◽  
Plasma Membrane Lipid ◽  
Cytoplasmic Vesicles ◽  
Immunocytochemical Techniques ◽  
Phosphatidylinositol 4

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.

scholarly journals Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases

1986 ◽  
Vol 240 (2) ◽  
pp. 341-348 ◽  
Author(s):  
A Imai ◽  
M J Rebecchi ◽  
M C Gershengorn
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Product Inhibition ◽  
Differential Regulation ◽  
Gh3 Cells ◽  
Maximal Effect ◽  
Phosphatidylinositol Kinase ◽  
Rat Pituitary ◽  
Phosphoinositide Kinase ◽  
Phosphatidylinositol 4

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.

scholarly journals Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat β-cell tumour

1984 ◽  
Vol 219 (2) ◽  
pp. 471-480 ◽  
Author(s):  
N E Tooke ◽  
C N Hales ◽  
J C Hutton
Keyword(s):  
Plasma Membrane ◽  
Beta Cell ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Secretory Granules ◽  
Cell Tumour ◽  
Density Gradients ◽  
Phosphatidylinositol Kinase ◽  
Phosphatidylinositol 4

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

scholarly journals Control of diverse subcellular processes by a single multi-functional lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]

2016 ◽  
Vol 473 (12) ◽  
pp. 1681-1692 ◽  
Author(s):  
Sourav Kolay ◽  
Urbashi Basu ◽  
Padinjat Raghu
Keyword(s):  
Plasma Membrane ◽  
Eukaryotic Cells ◽  
Head Group ◽  
Molecular Processes ◽  
Lipid Kinase ◽  
Multiple Functions ◽  
Lipid Species ◽  
Subcellular Processes ◽  
Invariant Functional ◽  
Standing Question

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a multi-functional lipid that regulates several essential subcellular processes in eukaryotic cells. In addition to its well-established function as a substrate for receptor-activated signalling at the plasma membrane (PM), it is now recognized that distinct PI(4,5)P2 pools are present at other organelle membranes. However, a long-standing question that remains unresolved is the mechanism by which a single lipid species, with an invariant functional head group, delivers numerous functions without loss of fidelity. In the present review, we summarize studies that have examined the molecular processes that shape the repertoire of PI(4,5)P2 pools in diverse eukaryotes. Collectively, these studies indicate a conserved role for lipid kinase isoforms in generating functionally distinct pools of PI(4,5)P2 in diverse metazoan species. The sophistication underlying the regulation of multiple functions by PI(4,5)P2 is also shaped by mechanisms that regulate its availability to enzymes involved in its metabolism as well as molecular processes that control its diffusion at nanoscales in the PM. Collectively, these mechanisms ensure the specificity of PI(4,5)P2 mediated signalling at eukaryotic membranes.

scholarly journals Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes

1987 ◽  
Vol 241 (3) ◽  
pp. 835-845 ◽  
Author(s):  
D E Whipps ◽  
A E Armston ◽  
H J Pryor ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Isolated Rat Hepatocytes ◽  
Guanine Nucleotide Regulatory Protein ◽  
Phosphatidylinositol 4

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.

scholarly journals Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes

1993 ◽  
Vol 290 (1) ◽  
pp. 179-183 ◽  
Author(s):  
O M Santiago ◽  
L I Rosenberg ◽  
M E Monaco
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Tumour Cells ◽  
Assay System ◽  
Mammary Tumour ◽  
Transferase Activity ◽  
Phosphoinositide Cycle

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.

scholarly journals Septin function tunes lipid kinase activity and phosphatidylinositol 4,5 bisphosphate turnover during G-protein coupled PLC signaling in vivo

2021 ◽  
Author(s):  
Aastha Kumari ◽  
Avishek Ghosh ◽  
Sourav Kolay ◽  
RAGHU PADINJAT
Keyword(s):  
Plasma Membrane ◽  
Kinase Activity ◽  
Electrical Response ◽  
Receptor Activation ◽  
Sensory Transduction ◽  
Photon Absorption ◽  
Lipid Kinase ◽  
Phosphatidylinositol 4

The hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2 depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2 is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K, dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. The activity of dPIP5KL in vitro is enhanced by depletion of PNUT, a non-redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of pnut rescues the effect of dPIP5KL depletion on the light response and PI(4,5)P2 resynthesis during PLC signalling. Lastly we find that depletion of Septin Interacting Protein 1 (Sip1),previously shown to bind PNUT, phenocopies the effect of dPIP5KL depletion in vivo. Thus, our work defines a septin 7 and Sip1 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

scholarly journals A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

2021 ◽  
Author(s):  
Hannes Maib ◽  
David H Murray
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Lipid Kinase ◽  
Phosphoinositide Signaling ◽  
Cargo Delivery ◽  
Membrane Tethering ◽  
Molecular Machinery ◽  
Protein Components ◽  
Phosphatidylinositol 4

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane.

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