Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling (original) (raw)
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The role of the phosphoinositides at the Golgi complex
Biochimica Et Biophysica Acta-molecular Cell Research, 2005
The phosphorylated derivatives of phosphatidylinositol (PtdIns), known as the polyphosphoinositides (PIs), represent key membranelocalized signals in the regulation of fundamental cell processes, such as membrane traffic and cytoskeleton remodelling. The reversible production of the PIs is catalyzed through the combined activities of a number of specific phosphoinositide phosphatases and kinases that can either act separately or in concert on all the possible combinations of the 3, 4, and 5 positions of the inositol ring. So far, seven distinct PI species have been identified in mammalian cells and named according to their site(s) of phosphorylation: PtdIns 3-phosphate (PI3P); PtdIns 4-phosphate (PI4P); PtdIns 5-phosphate (PI5P); PtdIns 3,4-bisphosphate (PI3,4P2); PtdIns 4,5-bisphosphate (PI4,5P2); PtdIns 3,5bisphosphate (PI3,5P2); and PtdIns 3,4,5-trisphosphate (PI3,4,5P3). Over the last decade, accumulating evidence has indicated that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated phosphoinositides, but also as regulators of different protein targets in their own right. These regulatory actions are mediated through the direct binding of their protein targets. In this way, the PIs can control the subcellular localization and activation of their various effectors, and thus execute a variety of cellular responses. To exert these functions, the metabolism of the PIs has to be finely regulated both in time and space, and this is achieved by controlling the subcellular distribution, regulation, and activation states of the enzymes involved in their synthesis and removal (kinases and phosphatases). These exist in many different isoforms, each of which appears to have a distinctive intracellular localization and regulation. As a consequence of this subcompartimentalized PI metabolism, a sort of ''PI-fingerprint'' of each cell membrane compartment is generated. When combined with the targeted recruitment of their protein effectors and the different intracellular distributions of other lipids and regulatory proteins (such as small GTPases), these factors can maintain and determine the identity of the cell organelles despite the extensive membrane flux [S. Munro, Organelle identity and the organization of membrane traffic, Nat. Cell. Biol. 6 (2004) 469 -4721]. Here, we provide an overview of the regulation and roles of different phosphoinositide kinases and phosphatases and their lipid products at the Golgi complex. D
Phosphoinositides, Major Actors in Membrane Trafficking and Lipid Signaling Pathways
Phosphoinositides are lipids involved in the vesicular transport of proteins and lipids between the different compartments of eukaryotic cells. They act by recruiting and/or activating effector proteins and thus are involved in regulating various cellular functions, such as vesicular budding, membrane fusion and cytoskeleton dynamics. Although detected in small concentrations in membranes, their role is essential to cell function, since imbalance in their concentrations is a hallmark of many cancers. Their synthesis involves phosphorylating/dephosphorylating positions D3, D4 and/or D5 of their inositol ring by specific lipid kinases and phosphatases. This process is tightly regulated and specific to the different intracellular membranes. Most enzymes involved in phosphoinositide synthesis are conserved between yeast and human, and their loss of function leads to severe diseases (cancer, myopathy, neuropathy and ciliopathy).
Molecular Biology of The Cell, 2005
Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate (PtdIns(3)P) on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P 2 ) binds the PH domain-containing proteins Akt and TAPP-1. Type Iα inositol polyphosphate 4phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P 2 forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4phosphatase co-localised with early and recycling endosomes. Upon growth factor stimulation 4-phosphatase endosomal localization persisted, but in addition the 4phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns3-P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts (MEFs) from homozygous Weeble mice, which have a mutation in the type I 4phosphatase, exhibited dilated early endosomes. 4-phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP-1 PH domain. The 4-phosphatase contains C2 domains which bound PtdIns(3,4)P 2 and C2-domain-deletion mutants lost PtdIns(3,4)P 2 4-phosphatase activity, did not localize to endosomes, or inhibit TAPP-1-PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates PI 3-kinase signals at distinct subcellular locations. asparagine and ligated into the KpnI site of pTrcHisC (pTrcHis-D693N). N-terminal GST-tandem C2 domains were PCR amplified and subcloned into the pGEX-KG HindIII site. 4-phosphatase (aa 305-939) was PCR amplified and subcloned into the KpnI site of pTrcHisC, or into the HindIII site of pGEX-KG (pTrcHis-4ptase∆C2AB and pGEX-KG-4ptase∆C2AB). Constructs were transformed into BL21 E.coli, incubated to an OD 600 0.6, and protein induced with isopropyl β-D-thiogalactopyranoside (1 mM final). Pelleted bacteria were resuspended in 50 mM Tris, pH 8, 1M NaCl, protease inhibitor cocktail (Roche) lysed by 3 x sonication 30 sec on ice, pelleted 19,000 x g (15 min) and purified by Ni 2+ affinity chromatography. Ni-NTA resin (2 ml) (Scientifix), pre-equilibrated with lysis buffer, was added to the cleared bacter ial lysate and rocked at 4°C 1 hr, pelleted at 700 x g for 5 min, washed, proteins eluted with 10 ml 75 mM imidazole, 50 mM Tris, pH 8, 1 M NaCl and concentrated using a Vivaspin 10,000 MW cut off (Vivascience). 2 ml glutathione Sepharose resin, pre-equilibrated with PBS, was added to the cleared bacterial lysate, rocked at 4°C for 1 hr, pelleted at 500 xg for 5 min, washed with ice cold PBS, and eluted with 10 mM glutathione, 50 mM Tris pH 8.
A phosphoinositide conversion mechanism for exit from endosomes
Nature, 2016
Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-...
Phosphoinositides: Regulators of membrane traffic and protein function
FEBS Letters, 2007
Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research.
Journal of Biological Chemistry, 2007
Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) is proposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P 2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P 2 subcellular distribution. PtdIns(4,5)P 2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in ⌬sac1 ⌬inp54 and sac1 ts ⌬inp54 mutants. Furthermore, sac1 ts ⌬inp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P 2 . Under these conditions PtdIns(4,5)P 2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P 2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P 2 . ⌬sac1 ⌬inp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P 2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P 2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.
Functional studies of the mammalian Sac1 phosphoinositide phosphatase
Advances in Enzyme Regulation, 2009
PIPs are phosphorylated derivatives of phosphatidylinositol (PtdIns) that serve primary intracellular roles: (i) in the specification of dedicated membrane microdomains that organize signal transduction processes, (ii) as co-factors for the regulated activities of proteins, and (iii) as precursors for second messengers such as diacylglycerol and soluble inositol phosphates . In part, this diversification of function reflects the chemical diversity afforded by the poly-hydroxylated inositol headgroup. Mammalian cells express seven distinct PIP species -phosphatidylinositol 3-phosphate (PtdIns-3-P), PtdIns-4-P, PtdIns-5-P, phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P 2 ), PtdIns-4,5-P 2 , PtdIns-3,4-P 2 , and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) -each of which interfaces with specific downstream effectors . Tight regulation of each of these PIP levels is essential to cellular processes that include vesicular trafficking, apoptosis, metabolism, actin reorganization, cell proliferation and cell growth . Yeast do not synthesize PtdIns-3,4-P 2 or PtdIns-3,4,5-P 3 and, indeed, 3-OH PIPs are nonessential for yeast viability although these discharge important homeostatic functions .
Modular phosphoinositide-binding domains – their role in signalling and membrane trafficking
Current Biology, 2001
The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.