Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling.
ABSTRACT: Phosphoinositides (PIs) have long been known to have important roles in cell signalling. During the past decade, it has become clear that these lipids also act as constitutive signals that aid in defining organelle identity, and are short-lived recruiters and regulators of cytoskeletal and membrane dynamics. Recent studies have provided important clues as to how regulated activation of PI-metabolizing enzymes and recruitment of their binding proteins might cooperate in targeting distinct pools of PIs to different cell physiological functions.
Project description:Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdInsP) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.
Project description:Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.
Project description:Phosphoinositides (PIs) are a group of key signaling and structural lipid molecules involved in a myriad of cellular processes. PI phosphatases, together with PI kinases, are responsible for the conversion of PIs between distinctive phosphorylation states. PI phosphatases are a large collection of enzymes that are evolved from at least two disparate ancestors. One group is distantly related to endonucleases, which apply divalent metal ions for phosphoryl transfer. The other group is related to protein tyrosine phosphatases, which contain a highly conserved active site motif Cys-X5-Arg (CX5R). In this review, we focus on structural insights to illustrate current understandings of the molecular mechanisms of each PI phosphatase family, with emphasis on their structural basis for substrate specificity determinants and catalytic mechanisms. This article is part of a Special Issue entitled Phosphoinositides.
Project description:Multiple TRP channels are regulated by phosphoinositides (PIs). However, it is not known whether PIs bind directly to TRP channels. Furthermore, the mechanisms through which PIs regulate TRP channels are obscure. To analyze the role of PI/TRP interactions, we used a biochemical approach, focusing on TRPC6. TRPC6 bound directly to PIs, and with highest potency to phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). We found that PIP(3) binding disrupted the association of calmodulin (CaM) with TRPC6. We identified the PIP(3)-binding site and found that mutations that increased or decreased the affinity of the PIP(3)/TRPC6 interaction enhanced or reduced the TRPC6-dependent current, respectively. PI-mediated disruption of CaM binding appears to be a theme that applies to other TRP channels, such as TRPV1, as well as to the voltage-gated channels KCNQ1 and Ca(v)1.2. We propose that regulation of CaM binding by PIs provides a mode for integration of channel regulation by Ca(2+) and PIs.
Project description:Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction.
Project description:Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.
Project description:Phosphoinositides (PIs) play pivotal roles in the regulation of many biological processes. The quality and quantity of PIs is regulated in time and space by the activity of PI kinases and PI phosphatases. The number of PI-metabolizing enzymes exceeds the number of PIs with, in many cases, more than one enzyme controlling the same biochemical step. This would suggest that the PI system has an intrinsic ability to buffer and compensate for the absence of a specific enzymatic activity. However, there are several examples of severe inherited human diseases caused by mutations in one of the PI enzymes, although other enzymes with the same activity are fully functional. The kidney depends strictly on PIs for physiological processes, such as cell polarization, filtration, solute reabsorption, and signal transduction. Indeed, alteration of the PI system in the kidney very often results in pathological conditions, both inherited and acquired. Most of the knowledge of the roles that PIs play in the kidney comes from the study of KO animal models for genes encoding PI enzymes and from the study of human genetic diseases, such as Lowe syndrome/Dent disease 2 and Joubert syndrome, caused by mutations in the genes encoding the PI phosphatases, OCRL and INPP5E, respectively.
Project description:Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.
Project description:Despite being a minor component of cells, phosphoinositides are essential for eukaryotic membrane biology, serving as markers of organelle identity and involved in several signaling cascades. Their many functions, combined with alternative synthesis pathways, make in vivo study very difficult. In vitro studies are limited by their inability to fully recapitulate the complexities of membranes in living cells. We engineered the biosynthetic pathway for the most abundant phosphoinositides into the bacterium Escherichia coli, which is naturally devoid of this class of phospholipids. These modified E. coli, when grown in the presence of myo-inositol, incorporate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI4P), phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylinositol-3,4,5-trisphosphate (PIP3) into their plasma membrane. We tested models of biophysical mechanisms with these phosphoinositides in a living membrane, using our system to evaluate the role of PIP2 in nonconventional protein export of human basic fibroblast growth factor 2. We found that PI alone is sufficient for the process.
Project description:It is well established that the spatial- and temporal-restricted generation and turnover of phosphoinositides (PIs) by a cascade of PI-metabolizing enzymes is a key regulatory mechanism in the endocytic pathway. Here, we demonstrate that the Sac1 domain-containing protein Sac2 is a PI 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate in vitro. We further show that Sac2 colocalizes with early endosomal markers and is recruited to transferrin (Tfn)-containing vesicles during endocytic recycling. Exogenous expression of the catalytically inactive mutant Sac2C458S resulted in altered cellular distribution of Tfn receptors and delayed Tfn recycling. Furthermore, genomic ablation of Sac2 caused a similar perturbation on Tfn and integrin recycling as well as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway.