have shown that in sciatic nerve endoneurial preparations from diabetic rats, the protein kinase C agonists phorbol myristate acetate and dioctanoylglycerol ...
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32 I
Greene, D. A,, De Jesus, P. V. & Winegrad, A. I. (1975) J. Clin. Invest. 55, 132&1336. Harwood, J. L. & Hawthorne, J. N. (1969) Biochim. Biophys. Acra 171,75-88 Hawthorne, J. N. (1985)Proc. Nufr. Soc. 44, 167 172 Hawthorne, J. N. (1986)In/. Rev. Neurohiol. 28,241 273 Hokin, M. R. & Hokin, L. E. (1953)J . B i d . Chem. 203,967-977 Irvine, R. F.,Letcher, A. J. & Dawson, R. M . C. (1984)Biochem. J . 218. 177-185 Kaplan, D. R., Whitman, M., Schamausen, B., Raptis. L., Garcea. R. L.. Pallas, D., Roberts, T. M . & Cantley, L. (1986)Proc. Nut/. Acud. Sci. U . S . A . 83,3624-3628 Kemp. P., Hubscher, G. & Hawthorne, J. N . (1959)Biochim. Biophys. Acta 31, 585-586 Kemp, P., Hubscher. G. & Hawthorne, J. N. (1961)Biochern. J . 79, 193 200 Kirk, C. J., Creba, J. A,. Downes, C. P. & Michell, R. H. (1981) Biochem. Soc. Truns. 9. 377 379 Knowles, A. F.(1986)Arch. Biochem. Biophys. 249. 76 87 Levebre, Y.A., White, D. A. & Hawthorne, J. N. (1976) Can. J . Biochem. 54. 746 753 Low, M. G., Ferguson, M. A. G., Futerman, A. H. & Silman. I . (1986)Trends Biuchern. Sci. 11, 212-215 Macara. I . G., Marinetti. G. V. & Balduzzi, P. C. (1984)Pro(,. Null. Acud. Sei. U.S.A. 81, 2728~-2732 MacDonald, M . L.. Kvenzel, E. A., Glomset, J. A. & Krebs, E. G. (1985)Proc. Null. Acad. Sci. U . S . A . 82. 3993-3997 Our phosphoinositide studies have been supported by the Wellcome Mayhew, J. A,, Gillon, K. R. W. & Hawthorne, J . N. (1983) Trust. the Medical Research Council, the British Heart Foundation Diahetologia 24. 13-1 5 (S.H.S.), Pfizer U.K. Ltd. (C.M.F.S.) and the Japan Society for the Merritt, J. A,. Taylor. C. W.. Rubin. R. P. & Putncy, J. W.. Jr. f 1986) Promotion of Science (H.Y.) Biodieni. J . 236. 337 343 Michell, R. H. (1975)Biochim. Biophys. Acta 415,81 -147 Palmano. K . P., Whiting, P. H . & Hawthorne. J. N. (1977)Biochrm. Abdel-Latif. A. A,. Akhtar, R. A. & Hawthorne, J. N. (1977) BioJ . 167,229-235 chrm. J . 162. 61 73 Rodnight, R. (1956)Biochem. J . 63,223 231 Berridge. M. J. (1983)Biochem. J . 212. 849-858 Saltiel, A. R. & Cuatrecasas, P. (1986)Proe. Narl. Acad. Sci. U.S.A. Berridge, M. J. (1984)Biochrm. J . 220,345-360 83,5793-5797 Berti-Mattere. L.. Petersen. R.. Bell, M. & Eichberg, J. (1985) J. Simpson, C. M. F. & Hawthorne, J. N. (1986)Biochem. Soc. Trans. Ncwrochrm. 45. 1692 1698 14,364 -365 Cockroft. S. & Gomperts, B. D. (1985)Nature (London) 314,534-536 Sugano, S. & Hanafusa, H. (1985)Mol. Cell. Biol. 5,2399 -2404 Crcha. J. A.. Downes. C. P.. Hawkins. P. T., Brewster, G., Michell. R. H. Sugimoto, Y . & Erikson, R. L. (1985)Mol. Cell. B i d . 5, 3194 3198 & Kirk, C . J. (19x3) Bioc.hcwi. J . 212. 733 747 Sugimoto, Y.,Whitman, M., Cantley, L. C. & Erikson, R. L. (1984) Dawson. R. M. C. (1954)Biochem. J . 57, 237-245 Proc. Null. Acad. Sci. U.S.A. 81,21 17-2121 Durell. J.. Sodd, M. A. & Friedel, R. 0. (1968)Life Sci. 7, 363-368 Wallace, M . A. & Fain. J. N. (1985)J . Biol. Chrm. 260,9527 9530 Fry, M. J.. Gebhardt. A.. Parker. P. J. & Foulkes, J. G. (1985) Whitman. M., Kaplan, D., Cantley, L., Roberts, T. M. & SchaffEMBO J . 4. 3173 3178 hausen, B. (1986)Fed. Proc. Fed. Am. Soe. Exp. B i d . 45.2647 2652 Gillon. K. R. W. & Hawthorne. J. N . (1983)Biochem. J . 210,775 781 Wilson, D. B., Bross, T. E., Hofmann, S. L. & Majerus, P. W. (1984) Gillon. K. R. W.. Hawthorne, J. N. & Tomlinson, D. R. (1983) J . B i d . Chem. 259. 11718-1 I724 Diuhc~tologia25. 365 37 I Greene. D. A. & Lattimer, S. A. (1983)J. Clin. Invest. 72, 1058-1063 Received 1 December 1986 Greenc, D. A. & Lattimer. S. A. (1986)Diaheres 35, 242-245
This work suggests that there is a link between inositol and the Na/K+ ATPase of nerve which could involve the phosphoinositides. Berti-Mattere et al. ( 1985) have shown that "P-labelling of the polyphosphoinositides is increased in nerves from diabetic animals and that insulin reverses the change. Nerve conduction is associated with increased labelling of these lipids (reviewed by Hawthorne, 1986) but the reasons for this are not clear. Greene & Lattimer (1986) have shown that in sciatic nerve endoneurial preparations from diabetic rats, the protein kinase C agonists phorbol myristate acetate and dioctanoylglycerol increased the rate of ouabain-inhibitable oxygen consumption, a parameter taken to reflect Na' / K + ATPase activity, to a level comparable with that of control nerves. It might follow that kinase C is less active in nerves from diabetic animals, but as yet there is no direct evidence for this. Nor d o we know whether the activity of the N a + / K + ATPase is modulated by kinase C phosphorylation. Since inositol transport into nerve is Na+ -linked (Gillon & Hawthorne, 1983), an inositol requirement for the sodium pump will lead to a self-reinforcing cycle of damage. Further information about this system could lead to clinical advances in the management of diabetic neuropathy.
Phosphatidylinositol-transferprotein and cellular phosphatidylinositol metabolism PETER A. VAN PARIDON,* PENTTI SOMERHARJUt and KAREL W. A. WlRTZ*$ * Laboratory of Biochemistry, State University of Utrecht, Padualuan 8 , 3584 C H Utrecht, The Netherlands, and, 7 DcJpartmcnt of Basic Chemistry, University of Helsinki, Siltavuorcwpenger 1 0 , SF-Helsinki 17, Finland All mammalian tissues thus far examined contain the phosphatidylinositol-transfer protein (PI-TP) (for reviews, see Wirtz, 1982; Helmkamp, 1985). This protein binds phosphatidylinositol (PI) and transfers it between membranes (Demel c't al., 1985). By this mode PI-TP may be one of the Abbreviations used: PI-TP. phosphatidylinositol-transfer protein; PI, phosphatidylinositol; PC. phosphatidylcholine; PG, phosphatidylglycerol: PA. phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethaiiolaminc: PIP. phosphalidylinositol4-phosphate;PIP,, phosphatidylinositol 4.5-bisphosphate. $To whom correspondence should be addressed. VOl.
I5
vehicles involved in the intracellular transfer of PI to membranes in need of a constant supply of PI (e.g. the plasma membrane) (Michell, 1975; Hokin, 1985). PI-TP has been purified to homogeneity from bovine brain (Helmkamp et al., 1974, 1976; Demel et al., 1977) and heart (DiCorleto r t al., 1979) and human platelets (George & Helmkamp, 1985). A partial purification of PI-TP was reported from rat liver (Lumb et al., 1976) and porcine platelets (Laffont et af., 1981). This protein also occurs in yeast (Daum & Paltauf, 1984). An intriguing aspect of PI-TP is that it is not monospecific for PI but also transfers phosphatidylcholine (PC) and phosphatidylglycerol (PG), although at a significantly lower rate (Kasper & Helmkemp, 1981; Somerharju el al., 1983). Phospholipids like phosphatidic acid (PA), phosphatidylserine (PS) and phosphatidylethanolamine (PE) are not transferred by PI-TP (DiCorleto et al., 1979; Zborowski & Demel, 1982). The question that we address in this report, is the peculiar combination of phospholipids bound and transferred by PI-TP and the possibility that this spectrum of affinities has
BIOCHEMICAL SOCIETY TRANSACTIONS
322 a particular physiological significance. Here we present some recent data in support of the hypothesis that the interplay between PI-TP and a membrane may control the relative amounts of PI and PC in that membrane. In addition, we will refer to PI-TP as a tool to introduce PI into intact cells. Preferential binding
of PI
Isolation of PI-TP from bovine brain yielded two subforms, identical in molecular mass (i.e. 35 kDa) but different in isoelectric point (i.e. pl 5.5 for PI-TP I and pl 5.7 for PI-TP I I ) (Van Paridon et al., 1987; see also Helmkamp et al., 1976). A similar feature was observed for PI-TP from bovine heart (DiCorleto e t al., 1979) and from human platelets (George & Helmkamp, 1985). We have recently demonstratcd that this difference in charge is due to the fact that PI-TP I contains one molecule of PI and PI-TP 11 one molecule of PC. Both subforms are interconvertible by incubation with vesicles consisting of the alternate phospholipid. With respect to the fact that bovine brain contains approximately ten times more PC than PI, it is of interest that upon isolation from this tissue 65% of PI-TP is of the subform I and 35% of the subform 11. This is already a clear indication that PI-TP preferentially binds PI. Injection of PI-TP under a phospholipid monolayer spread from an equimolar mixture of ['4C]PC/PI or of PC/['4C]PI at the air-water interface demonstrated that PI-TP bound PI ten times faster than PC (Demel e t al., 1977). By remaining constant the surface pressure indicated that the extraction of a PI or PC molecule from the monolayer was accompanied by the insertion of a phospholipid molecule initially bound to PI-TP, i.e. the binding reflects an exchange mechanism. I n some recent studies binding to PI-TP was measured spectrofluorimetrically by the use of PI and PC carrying a pyrene-labelled Fatty acid at the sn-2 position. Vesicles of these phospholipids and an internal quencher (i.e. 10 mol YO trinitrophenyl-PE) were prepared so that upon excitation at 346 nm no pyrenyl fluorescence was measurable at 378 nm. Titration of these quenched vesicles with PI-TP gave rise to fluorescence due to binding of pyrenyl-PI or pyrenyl-PC to the protein. The fluorescence yield increased linearly with
Pyr-PI
PljPC Pyr-PC
PCjPl
Vesicle composition
Fig. I . E f / i ~ . /o / unluhellid phospholipids on the binding of pJ'renyl-PI und pjwn,vl-PC 10 PI- T P
Binding, as detected by the increase of pyrene monomer fluorescence, was measured by titrating vesicles consisting of pyrenylPI ((I) or pyrenyl-PC ( h ) .various amounts ofegg PC or yeast PI, and an internal fluorescence quencher (i.e. IOmol% trinitrophenyl-PE) with PI-TP. Binding per p g of transfer protein was plotted as a function of increasing amounts of unlabelled lipids. The decrease in pyrenyl-phospholipid binding is a measure of the binding affinity of PI-TP for this labelled lipid relative to that for the unlabelled lipid. Abbreviation: Pyr, pyrenyl.
the amount of PI-TP and was a measure of the affinity of PI-TP for these phospholipids. Incorporation of increasing amounts of unlabelled PI or PC into the quenched vesicles affected the binding of pyrenyl-PI and pyrenyl-PC to PI-TP as a result of competition (Fig. 1). From the linear relationship between the mol YOof yeast PI present in the vesicles and the decrease of the binding of pyrenyl-P1, one can infer that PI-TP has approximately the same affinity for both PI species. A similar conclusion may be drawn for egg PC relative to pyrenyl-PC. Due to the low affinity of PI-TP for PC this phospholipid has relatively little effect on the binding of pyrenyl-PI (Fig. la); the opposite is true for the effect of yeast PI on the binding of pyrenyl-PC (Fig. Ih). Graphically one can estimate that the affinity of PI-TP for pyrenyl-PI relative to egg PC is 9.7, for pyrenyl-PC relative to yeast PI 0.07 and for yeast PI relative to egg PC 15.6. Thus under conditions where the phospholipid redistribution reaches an equilibrium between protein and vesicles, PI-TP binds PI 16 times more efficiently than PC. In the previous experiment 1-palmitoyl, 2-pyrenyloctanoyl-PI (C,, o , PyrC,-PI) was used. By varying the fatty acid at the sn-l position and the length of the fatty acid carrying the pyrene-moiety from 6 to 14 carbon atoms, we observed that PI-TP has the highest affinity for C,, I , PyrC,-PI and that the affinity decreased in the order C,,.,, PyrC,-PI > C,,:,, PyrC,-PI > C,, ,-PI = C,,., ,PyrC,-PI. The relatively low affinity of PI-TP for the PI species carrying the C,, fatty acids raises the question of how efficiently 1 -stearoyl, 2-arachidonoyl-PI, the most abundant species of PI in mammalian tissues, is bound by this protein. The search for the physiological role of PI-TP begs an answer to this question. Net transfer o f Pi and its regulation
PI-TP transfers PI from donor membranes (e.g. single bilayer vesicles, PI-monolayers) to acceptor vesicles consisting of PC but lacking PI (Demel et al., 1977; Kasper & Helmkamp, 1981). This net unidirectional transfer of PI is compensated by a flux of PC in the opposite direction. PI-TP will continue to exchange its bound PI molecule for a PC molecule from the vesicles until the ratio of PI and PC in the accepting vesicle matches that of the donor membrane. I t is of note that for both the endoplasmic reticulum and the plasma membrane of rat liver the content of PI and PC is such that interaction of PI-TP with these membranes results in the same relative amounts of PI and PC bound to PI-TP, i.e. 2.5. times more PI than PC. This implies that with an affinity ratio of 16 for PI over PC, PI-TP will instantaneously compensate for any depletion of PI in the plasma membrane as may occur during agonist-induced phosphoinositide metabolism (Hokin, 1985). In this model, with PI and PC being continuously synthesized on the endoplasmic reticulum, the interplay between PI-TP and the plasma membrane may well keep the relative amounts of these phospholipids in that membrane within narrow limits. This proposed mechanism of protein-mediated monomer phospholipid transfer could be complementary to the route by which phospholipids reach the plasma membrane by vesicular movement (Morre, 1977). It was recently shown in yeast that the supply of PI to the plasma membrane occurred by mechanisms other than secretory vesicles (Daum r t al., 1986). From model membrane studies it is known that PI-TP activity is strongly affected by the incorporation of acidic phospholipids (e.g. PA, PI, PS, PG) into vesicles (Somerharju et al., 1983; Yoshimura & Helmkamp, 1984). This effect seems to be due to relatively non-specific electrostatic interactions leading to an enhanced association with the vesicle surface. Recently, we have extended these studies by measuring the effect of phosphatidylinositol 4-phosphate 1987
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(PIP) and phosphatidylinositol 4,s-bisphosphate (PIP,) on a controlled manipulation of cellular PI may specifically the activity of PI-TP. In these experiments the acidic phos- affect malignant cells. Application of PI-TP in facilitating pholipids were added as a third component to the donor/ the introduction of defined molecular species of PI into cells acceptor vesicle assay system (Machida & Ohnishi, 1978). may help to advance our knowledge of how the metabolism Under standard assay conditions (i.e. 0.5 ~ M - Co,, , PyrC,,of PI affects the functioning of the cell. P c , ~ ~ ~ ~ M - P C 85: -PA 15mol , YO,0.014p~-PI-TP)halfmaximal inhibition of transfer was observed with 0 . 3 3 ~ ~ PIP and O.IO~M-PIP,as compared with 2 5 p ~ - P I .This Campbell, C. R., Fishman, J. B. & Fine, R. E. (1985) J . B i d . Chem. indicates that at this extent of inhibition the molar ratio of 260, I0948 I095 I PIPz over PI-TP was only seven-fold. The low ratio strongly Collins. C. A. & Wells, W. W. (1983) J. Biol. Chem. 258, 2130 2134 suggests that the inhibition is not caused by the binding of Daum, G. & Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385 391 Daum, G., Heidorn, E. & Paltauf, F. (1986) Biochim. Biophys. Acta PI-TP to a negatively charged interface, but is due to a direct 878, 93-101 affinity of PI-TP for PIP,. In this context it is worth noting Demel, R. A., Kalsbeek, R., Wirtz. K. W. A. & Van Deenen, L. L. M. that PI-TP does not transfer PIP (Schermoly & Helmkamp, (1977) Biochim. Biophw. Ac/u 466. 10-22 1983). At this point it is not clear whether PIP and PIP, play Demel, R. A,, Somerharju, P. & Wirtz, K. W. A. (1985) in Phosany particular role in the regulation of PI-TP in the cell. pho1ipid.Y in the Nervous System (Horrocks, L. A., Kanfer, J. N. & Porcellati, G . , eds.), vol. 2, pp. 61-70. Raven Press, New York Incorporation of' exogenous PI into cells DiCorleto, P. E., Warach, J. B. & Zilversmit, D. B. (1979) J . Biol. At present direct evidence is lacking that PI-TP is involved Chem. 254, 7795-7802 in the intracellular transport of PI from internal membranes George, P. Y. & Helmkamp, G. M. (1985) Biochim. Biophys. Acta836, 176-184 to the plasma membrane. On the other hand, PI-TP was Helmkamp, G. M. (1985) Chem. Phys. Lipids 38, 3 -16 shown to transfer PI from the sarcoplasmic reticulum to M.. Harvey, M. S.. Wirtz, K. W. A. & Van Deenen. sarcolemmal membranes in vitro (Shute et al., 1985). He1mkamp.G. L. L. M. (1974) J. B i d . Chem. 249. 6382-6389 Recently, we have demonstrated that this protein is very Helmkamp, G. M., Nelemans, S. A. & Wirtz, K. W. A. (1976) Bioeffective in introducing PI from vesicles into Friend erythrochim. Biophys. Acta 424, 168-182 leukaemic cells (Hohengasser et al., 1986). Incubation of Hohengasser, C . J. M., Thornburg, J. T., Van Paridon, P. A,, Van vesicles consisting of PE4'HlPI-cholesterol (46 :4 : 50mol YO; der Schaft, P. & Wirtz, K. W. A. (1986) J. B i d . Chem. 261. 0.5,umol of lipid) with cells and PI-TP (70pg) led to a 6255- 6259 time-dependent incorporation of ['HIPI (i.e. 10 nmol after Hokin, L. E. (1985) Annu. Rev. Biochem. 54, 205-235 4 h at 37°C). This protein-dependent incorporation was Jergil, B. & Sundler, R. (1983) J . Biol. Chem. 258, 7968 7973 Jett, M. & Alving, C. R. (1983) Biochem. Biophys. Res. Commun. 114. sup€rimposed on a spontaneous incorporation of ['HIPI 863-871 into these cells which proceeded at about one-third of the Jett, M., Chudzik, J., Alving, C. R. & Stanacev, N. Z. (1985) Cancer rate. Both in the absence and presence of PI-TP. ['HIPI was Res. 45. 4810-4815 incorporated to a similar extent into ['HIPIP and ['HIPIP, Kasper, A. M. & Helmkamp, G . M. (1981) Biochim. Biophys. Acta (i.e. up to 30% and 3%, respectively, of the [3H]PI spon664,22-32 taneously incorporated). These results strongly suggest that Laffont, F., Chap, H., Soula, G. & Douste-Blazy, L. (1981) Biochem. the ['HIPI incorporated by PI-TP did not become available Biophys. Res. Commun. 102. 1366~1371 Lumb, R. H., Kllosterman, A. D., Wirtz, K. W. A. & Van Deenen, for phosphorylation. It could be that PI, upon introduction L. L. M . (1976) Eur. J . Biochem. 69, 15-22 into the plasma membrane, is in equilibrium with the total cellular PI pool. It is also possible that this PI remains in the Machida, K. & Ohnishi, S. 1. (1978) Biochim. Biophys. Acta 507, 156-164 outer leaflet so that it does not become available for phosMichell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147 phorylation. The mode of uptake of the spontaneously Morre, D. J. (1977) Cell Surface Rev. 4, 1-83 incorporated PI is not known but, in view of the extensive Schermoly, M. J. & Helmkamp, G. M. (1983) Brain. Res. 268, 197 200 phosphorylation, it precludes a rapid equilibration with Shute, J. K., Van Paridon, P. A,, Smith, M. E. & Wirtz, K. W. A. endogenous PI pools. The preponderance of ['HIPIP for(1985) Biochem. Soc. Trans. ,413, 921-922 Somerharju, P., Van Paridon, P. & Wirtz, K. W. A. (1983) Biochim. mation suggests that this ['HIPI is lodged in a compartment Biophys. Acta 731, 186-195 which contains PI-kinase but lacks PIP-kinase. PI-kinase Van Paridon, P. A., Visser, A. J. W. G. & Wirtz, K. W. A. (1987) has been detected in lysosomes (Collins & Wells, 1983), the Biochim. Biophys. Aria in the press Golgi apparatus (Jergil & Sundler, 1983) and coated vesicles Wirtz, K. W. A. (1982) in Lipid-Protein Inreructions (Jost, P. C . & (Campbell et al., 1985) in support of the possibility that the Griffith, 0. H., eds.), vol. I , pp. 151L231, Wiley-Interscience, New spontaneous uptake of ['HIPI involves endocytosis. York In the study by Hohengasser et al. (1986) evidence was Yoshimura, T. & Helmkamp, G . M. (1984) Biochim. Biophys. Actu provided that the observed metabolism was restricted to PI 793, 463-470 species carrying arachidonic acid. In contrast to animal PI, Zborowski, J. & Demel, R. A. (1982) Biochim. Biophys. Acta 688, 381 387 spontaneous incorporation of plant PI into tumour cells in tissue culture was found to be cytotoxic (Jett & Alving, 1983). This difference in effect may reflect the presence of arachidonic acid in animal PI and its absence in plant PI Received 2 December 1986 (Jett e f al., 1985). These important observations suggest that -
Vol. 15
