Acyl chain selection couples the consumption and synthesis of phosphoinositides

Phosphoinositides (PIPn) in mammalian tissues are enriched in the stearoyl/arachidonoyl acyl chain species (“C38:4”), but its functional significance is unclear. We have used metabolic tracers (isotopologues of inositol, glucose and water) to study PIPn synthesis in cell lines in which this enrichment is preserved to differing relative extents. We show that PIs synthesised from glucose are initially enriched in shorter/more saturated acyl chains, but then rapidly remodelled towards the C38:4 species. PIs are also synthesised by a distinct ‘re‐cycling pathway’, which utilises existing precursors and exhibits substantial selectivity for the synthesis of C38:4‐PA and ‐PI. This re‐cycling pathway is rapidly stimulated during receptor activation of phospholipase‐C, both allowing the retention of the C38:4 backbone and the close coupling of PIPn consumption to its resynthesis, thus maintaining pool sizes. These results suggest that one property of the specific acyl chain composition of PIPn is that of a molecular code, to facilitate ‘metabolic channelling’ from PIP2 to PI via pools of intermediates (DG, PA and CDP‐DG) common to other lipid metabolic pathways.     

A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes

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.     

The uses and limitations of the analysis of cellular phosphoinositides by lipidomic and imaging methodologies

The advent of mass spectrometric methods has facilitated the determination of multiple molecular species of cellular lipid classes including the polyphosphoinositides, though to date methods to analyse and quantify each of the individual three PtdInsP and three PtdInsP₂ species are lacking. The use of imaging methods has allowed intracellular localization of the phosphoinositide classes but this methodology does not determine the acyl structures. The range of molecular species suggests a greater complexity in polyphosphoinositide signaling than yet defined but elucidating this will require further method development to be achieved. This article is part of a Special Issue entitled Tools to study lipid functions.     

Mapping the electrostatic profiles of cellular membranes

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately –35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Lipids and (glyco)proteins are the main constituents of biological membranes. Sugar moieties of glycoproteins, glycolipids, and adherent glycocalyx components such as hyaluronic acid can bear ionizable groups that confer a net negative charge on the outer surface of the plasma membrane. The aggregate surface charge of the outer membrane has been estimated indirectly by measuring the ζ potential—the potential at the slipping plane—by electrophoretic means (e.g., Tippe, 1981; Silva Filho et al., 1987) or by measuring streaming potentials (Vandrangi et al., 2012). The plasma membrane, however, is highly asymmetric; its inner (cytosolic) aspect is virtually devoid of carbohydrate moieties. Nevertheless, the cytosolic leaflet is also thought to be negatively charged, due primarily to the accumulation of anionic phospholipids, namely phosphoinositides and phosphatidylserine (PtdSer). Based on biochemical determinations of its lipid composition, the net negative charge of the plasmalemmal inner leaflet is estimated to generate an electrical field of 10⁵ V/cm (Olivotto et al., 1996). The membranes of intracellular organelles can also contain anionic lipids, but their precise lipid composition and topology have been difficult to assess and hence their surface charge has not been estimated.The surface potentials of biological membranes have important functional implications: they can alter the disposition of charged regions of transmembrane proteins, cause local accumulation of soluble counterions in the vicinity—altering the local pH as well as the concentration of physiologically important ions such as calcium—and serve to recruit extrinsic cationic proteins (McLaughlin, 1989). It is therefore important to determine the electrostatic properties of each of the organellar membranes. In principle, this could be accomplished by measuring the ζ potentials of isolated organelles. However, the purity of such preparations is imperfect, changes in lipid composition (particularly phosphoinositide degradation) and sidedness cannot be avoided, and loosely adherent components that may alter the surface charge can be removed during the isolation process. Alternative approaches to estimating the surface potential are therefore required.Here we used recombinant and synthetic polycationic peptides to obtain a quantitative estimate of the surface potential of the inner leaflet of the plasma membrane and to establish a hierarchical map of the potentials of the cytosolic surfaces of the major intracellular organelles in live cells.     

Phosphate number and acyl chain length determine the subcellular location and lateral mobility of phosphoinositides

Phosphoinositides are critical regulators of ion channel and transporter activity. There are multiple isomers of biologically active phosphoinositides in the plasma membrane and the different lipid species are non-randomly distributed. However, the mechanism by which cells impose selectivity and directionality on lipid movements and so generate a non-random lipid distribution remains unclear. In the present study we investigated which structural elements of phosphoinositides are responsible for their subcellular location and movement. We incubated phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with short or long acyl chains in CHO and HEK cells. We show that phosphate number and acyl chain length determine cellular location and translocation movement. In CHO cells, PI(4,5)P2 with a long acyl chain was released into the cytosol easily because of a low partition coefficient whereas long chain PI was released more slowly because of a high partition coefficient. In HEK cells, the cellular location and translocation movement of PI were similar to those of PI in CHO cells, whereas those of PI(4,5)P2 were different; some mechanism restricted the translocation movement of PI(4,5)P2, and this is in good agreement with the extremely low lateral diffusion of PI(4,5)P2. In contrast to the dependence on the number of phosphates of the phospholipid head group of long acyl chain analogs, short acyl chain phospholipids easily undergo translocation movement regardless of cell type and number of phosphates in the lipid headgroup.     

Thin-layer chromatography of the phosphoinositides

Thin-layer chromatography for the separation of mono-, di-, and triphosphoinositides (0.3-3 micro g total phosphorus) is described.     

Analysis of phosphoinositides in protein trafficking

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena. The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past. These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells.     

Mitochondrial respiratory chain adjustment to cellular energy demand

Because adaptation to physiological changes in cellular energy demand is a crucial imperative for life, mitochondrial oxidative phosphorylation is tightly controlled by ATP consumption. Nevertheless, the mechanisms permitting such large variations in ATP synthesis capacity, as well as the consequence on the overall efficiency of oxidative phosphorylation, are not known. By investigating several physiological models in vivo in rats (hyper- and hypothyroidism, polyunsaturated fatty acid deficiency, and chronic ethanol intoxication) we found that the increase in hepatocyte respiration (from 9.8 to 22.7 nmol of O(2)/min/mg dry cells) was tightly correlated with total mitochondrial cytochrome content, expressed both per mg dry cells or per mg mitochondrial protein. Moreover, this increase in total cytochrome content was accompanied by an increase in the respective proportion of cytochrome oxidase; while total cytochrome content increased 2-fold (from 0.341 +/- 0.021 to 0.821 +/- 0.024 nmol/mg protein), cytochrome oxidase increased 10-fold (from 0.020 +/- 0.002 to 0.224 +/- 0.006 nmol/mg protein). This modification was associated with a decrease in the overall efficiency of the respiratory chain. Since cytochrome oxidase is well recognized for slippage between redox reactions and proton pumping, we suggest that this dramatic increase in cytochrome oxidase is responsible for the decrease in the overall efficiency of respiratory chain and, in turn, of ATP synthesis yield, linked to the adaptive increase in oxidative phosphorylation capacity.     

Endosomal phosphoinositides and human diseases

Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3 P and PtdIns(3,5) P ₂, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as 'endosomal PIs'. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3 P and PtdIns(3,5) P ₂ 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdIns(3,5) P ₂ 5-phosphatase SAC3/FIG4 are implicated in Charcot–Marie–Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3 P 5-kinase PIP5K3/PIKfyve have been found in patients affected with François–Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.     

Mechanisms of cellular uptake of long chain free fatty acids

Cells take up long chain free fatty acids (FFA) in vivo from the non-protein bound ligand pools in extracellular fluid and plasma, which contain ~100 and 600 M albumin, respectively. The physiologic range of unbound FFA concentrations in such fluids has traditionally been calculated at < 1 M. Studies of [3H]-oleate uptake by hepatocytes, adipocytes, cardiac myocytes and other cell types demonstrate that FFA uptake within this range is saturable, and exhibits many other kinetic properties indicative of facilitated transport. Within this range, the uptake kinetics of the acidic (pKa = 0.5) FFA analog 2,2,3- heptafluorostearate are similar to those of stearate. Thus, uptake of physiologic concentrations of FFA involves facilitated transport of the FFA anion (FA-). Over a much wider range of unbound FFA concentrations hepatocellular [3H]-oleate uptake exhibits both saturable and non-saturable components. Oleate binding to liver plasma membranes (LPM) also demonstrates such components. Comparing the two components of FFA uptake to the corresponding components of binding permits estimates of trans-membrane transport rates. T1/2 for saturable uptake (~ 1 sec) is less than for non-saturable uptake (~ 14 sec). Others have determined the flip-flop rates of protonated FFA (FAH) across small and large unilamellar vesicles (SUV, LUV) and across cellular plasma membranes. These reported flip-flop rates, measured by the decrease in pH resulting from the accompanying proton flux, exhibit a highly significant inverse correlation with cell and vesicle diameter (r = 0.99). Although T1/2's in vesicles are in the msec range, those in cells are > 10 sec, and thus comparable to the rates of non-saturable uptake we determined. Thus, under physiologic conditions, the predominant mechanism of cellular FFA uptake is facilitated transport of FA-; at much higher, non- physiologic FFA concentrations, passive flip-flop of FAH predominates. Several plasma membrane proteins have been identified as potential mediators of facilitated FFA transport. Studies in animal models of obesity and non-insulin dependent diabetes mellitus demonstrate that tissue-specific regulation of facilitated FFA transport has important pathophysiologic consequences.     

The cellular localization of long chain fatty acids in sponges

Examination of fractionated sponge tissue shows that long chain fatty acids (LCFAs) occur in high proportions in cell membranes. This conclusion refutes a recent suggestion made by other workers that sponge membranes would contain conventional fatty acids similar to those found in membranes from other organisms.