Mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery

Experimental Cell Research

Volumn 247, Issue 1, 1999, Pages 61-78

  Préville, Xavier ^a   Salvemini, Francesca ^b   Giraud, Stéphane ^a   Chaufour, Sylvain ^a   Paul, Catherine ^a   Stepien, Georges ^a   Ursini, Matilde Valeria ^b   Arrigo, André Patrick ^a  

^a CENTRE DE GÉNÉTIQUE MOLÉCULAIRE ET CELLULAIRE   (France)

^b INSTITUTE OF GENETICS AND BIOPHYSICS ADRIANO BUZZATI TRAVERSO   (Italy)

Author keywords

Glucose 6 phosphate dehydrogenase; Glutathione; Glutathione peroxidase; Oxidative stress; Small heat shock proteins

Indexed keywords

GLUCOSE 6 PHOSPHATE DEHYDROGENASE; GLUTATHIONE; GLUTATHIONE PEROXIDASE; GLUTATHIONE REDUCTASE; GLUTATHIONE TRANSFERASE; HEAT SHOCK PROTEIN; HYDROGEN PEROXIDE;

ANIMAL CELL; ARTICLE; CELL DEATH; CELL MEMBRANE POTENTIAL; CELL STRUCTURE; CONTROLLED STUDY; ENZYME ACTIVITY; HUMAN; HUMAN CELL; MITOCHONDRIAL MEMBRANE; MOUSE; NONHUMAN; OXIDATION; OXIDATION REDUCTION REACTION; OXIDATIVE STRESS; PRIORITY JOURNAL; PROTEIN EXPRESSION;

ANIMALIA; MAMMALIA; MURINAE;

EID: 0345410965     PISSN: 00144827     EISSN: None     Source Type: Journal    
DOI: 10.1006/excr.1998.4347     Document Type: Article

References (105)

1. Davies K. J. A. Oxidative stress: The paradox of aerobic life. Free Radicals and Oxidative Stress: Environment, Drugs and Food Additives. 1995;Portland Press, London.
2. Jacobson M. D. Reactive oxygen species and programmed cell death. Trends Biochem. Sci. 21:1996;83-86.
3. Stadtman E. R. Protein oxidation and aging. Science. 257:1992;1220-1224.
4. Halliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis. 23:1993;118-126.
5. Meister A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal: Applications in research and therapy. Pharmacol. Ther. 51:1991;155-194.
6. Ciriolo M. R., Desideri A., Paci M., Rotilio G. Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I) · glutathione complex. J. Biol. Chem. 265:1990;11030-11034.
7. Serbinova E., Khwaja S., Rexnick A. Z., Packer L. Thioctic acid protects against ischemia-reperfusion injury in the isolated perfused Langendorff heart. Free Radical Res. Commun. 17:1992;49-58.
8. Winterbourn C. C. Superoxide as an intracellular radical sink. Free Radical Biol. Med. 14:1993;85-90.
9. Meister A., Anderson M. E. Glutathione. Annu. Rev. Biochem. 52:1983;711-760.
10. Pandolfi P. P., Sonati F., Rivi R., Mason P., Grosveld F., Luzzatto L. Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J. 14:1995;5209-5215.
11. Banki K., Hutter E., Colombo E., Gonchoroff N. J., Perl A. Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J. Biol. Chem. 271:1996;32994-33001.
12. Kuo W. Y., Tang T. K. Effects of G6PD overexpression in NIH3T3 cells treated with tert-butyl hydroperoxide or paraquat. Free Radical Biol. Med. 24:1998;1130-1138.
13. Macho A., Hirsch T., Marzo I., Marchetti P., Dallaporta B., Susin S. A., Zamzami N., Kroemer G. Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J. Immunol. 158:1997;4612-4619.
14. Zucker B., Bauer G. Intercellular induction of apoptosis of transformed cells is modulated by their intracellular glutathione concentration. Int. J. Oncol. 10:1997;141-146.
15. Zucker B., Hanusch J., Bauer G. Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species. Cell Death Differ. 4:1997;388-395.
16. Marchetti P., Castedo M., Susin S. A., Zamzami N., Hirsch T., Macho A., Haeffner A., Hirsch F., Geuskens M., Kroemer G. Mitochondrial permeability transition is a central coordinating event of apoptosis. J. Exp. Med. 184:1996;1155-1160.
17. Marchetti P., Decaudin D., Macho A., Zamzami N., Hirsch T., Susin S. A., Kroemer G. Redox regulation of apoptosis: Impact of thiol oxidation status on mitochondrial function. Eur. J. Immunol. 27:1997;289-296.
18. Zamzami N., Marchetti P., Castedo M., Decaudin D., Macho A., Hirsch T., Susin S. A., Petit P. X., Mignotte B., Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182:1995;367-377.
19. Zamzami N., Marchetti P., Castedo M., Zanin C., Vayssiere J. L., Petit P. X., Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181:1995;1661-1672.
20. Zamzami N., Susin S. A., Marchetti P., Hirsch T., Gomez-Monterrey I., Castedo M., Kroemer G. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183:1996;1533-1544.
21. Arrigo A.-P., Landry J. Expression and function of the low-molecular-weight heat shock proteins. Morimoto R. I., Tissieres A., Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. 1994;Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
22. Arrigo A.-P., Ahmad-Zadeh C. Immunofluorescence localization of a small heat shock protein (hsp 23) in salivary gland cells ofDrosophila melanogaster. Mol. Gen. Genet. 184:1981;73-79.
23. Arrigo A.-P., Suhan J. P., Welch W. J. Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein. Mol. Cell. Biol. 8:1988;5059-5071.
24. Arrigo A.-P., Welch W. Characterization and purification of the small 28,000-dalton mammalian heat shock protein. J. Biol. Chem. 262:1987;15359-15369.
25. Mehlen P., Mehlen A., Guillet D., Préville X., Arrigo A.-P. Tumor necrosis factor-α induces changes in the phosphorylation, cellular localization, and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine. J. Cell. Biochem. 58:1995;248-259.
26. Spector A., Li L. K., Augusteyn R. C., Schneider A., Freund T. α-Crystallin: The isolation and characterization of distinct macromolecular fractions. Biochem. J. 124:1971;337-343.
27. Arrigo A.-P. Tumor necrosis factor induces the rapid phosphorylation of the mammalian heat shock protein hsp28. Mol. Cell. Biol. 10:1990;1276-1280.
28. Crête P., Landry J. Induction of HSP27 phosphorylation and thermoresistance in Chinese hamster cells by arsenite, cycloheximide, A23187 and EGTA. Radiat. Res. 121:1990;320-327.
29. Kaur P., Saklatvala J. Interleukin 1 and tumour necrosis factor increase phosphorylation of fibroblast proteins. FEBS Lett. 241:1988;6-10.
30. Landry J., Lambert H., Zhou M., Lavoie J. N., Hickey E., Weber L. A., Anderson C. W. Human HSP 27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. J. Biol. Chem. 267:1992;794-803.
31. Mendelsohn M. E., Zhu Y., O'Neill S. The 29kDa proteins phosphorylated in thrombin activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein. Proc. Natl. Acad. Sci. USA. 88:1991;11212-11216.
32. Spector N. L., Mehlen P., Ryan C., Hardy L., Samson W., Levine H., Nadler L. M., Fabre N., Arrigo A.-P. Regulation of the 28kDa heat shock protein by retinoic acid during differentiation of human leukemic HL-60 cells. FEBS Lett. 337:1994;184-188.
33. Welch W. J. Phorbol ester, calcium ionophore, or serum added to quiescent rat embryo fibroblasts cells all result in the elevated phosphorylation of two 28,000-dalton mammalian stress proteins. J. Biol. Chem. 260:1985;3058-3062.
34. Kato K., Hasegawa K., Goto S., Inaguma Y. Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. J. Biol. Chem. 269:1994;11274-11278.
35. Mehlen P., Arrigo A.-P. The serum-induced phosphorylation of mammalian hsp27 correlates with changes in its intracellular localization and levels of oligomerization. Eur. J. Biochem. 221:1994;327-334.
36. Préville X., Schultz H., Knauf U., Gaestel M., Arrigo A. P. Analysis of the role of Hsp25 phosphorylation reveals the importance of the oligomerization state of this small heat shock protein in its protective function against TNFalpha- and hydrogen peroxide-induced cell death. J. Cell. Biochem. 69:1998;436-452.
37. Mehlen P., Hickey E., Weber L., Arrigo A.-P. Large unphosphorylated aggregates as the active form of hsp27 which controls intracellular reactive oxygen species and glutathione levels and generates a protection against TNFα in NIH-3T3-ras cells. Biochem. Biophys. Res. Commun. 241:1997;187-192.
38. Knauf U., Jakob U., Engel K., Buchner J., Gaestel M. Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance. EMBO J. 13:1994;54-60.
39. Lavoie J. N., Gingras-Breton G., Tanguay R. M., Landry J. Induction of chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. J. Biol. Chem. 268:1993;3420-3429.
40. Fuqua S. A. W., Oesterreich S., Hilsenbeck S. G., Von Hoff D. D., Eckardt J., Osborne C. K. Heat shock proteins and drug resistance. Breast Cancer Res. Treat. 32:1994;67-71.
41. Garrido C., Mehlen P., Fromentin A., Hammann A., Assem M., Arrigo A.-P., Chauffert B. Inconstant association between 27-kDa heat-shock protein (Hsp27) content and doxorubicin resistance in human colon cancer cells. The doxorubicin-protecting effect of Hsp27. Eur. J. Biochem. 237:1996;653-659.
42. Oesterreich S., Weng C.-N., Qiu M., Hilsenbeck S. G., Osborne C. K., Fuqua S. W. The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines. Cancer Res. 53:1993;4443-4448.
43. Richards E. H., Hickey E., Weber L. A., Master J. R. Effect of overexpression of the small heat shock protein HSP27 on the heat and drug sensitivities of human testis tumor cells. Cancer Res. 56:1996;2446-2451.
44. Arrigo A.-P. Small stress proteins: Chaperones that act as regulators of intracellular redox and programmed cell death. Biol. Chem. 379:1998;19-26.
45. Mehlen P., Schulze-Osthoff K., Arrigo A.-P. Small stress proteins as novel regulators of apoptosis - Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J. Biol. Chem. 271:1996;16510-16514.
46. Samali A., Cotter T. G. Heat shock proteins increase resistance to apoptosis. Exp. Cell Res. 223:1996;163-170.
47. Guenal I., Sidoti-de Fraisse C., Gaumer S., Mignotte B. Bcl-2 and Hsp27 act at different levels to suppress programmed cell death. Oncogene. 15:1997;347-360.
48. Arrigo A.-P. Expression of stress genes during development. Neuropathol. Appl. Neurobiol. 21:1995;488-491.
49. Mehlen P., Mehlen A., Godet J., Arrigo A.-P. hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells. J. Biol. Chem. 272:1997;31657-31665.
50. Huot J., Houle F., Spitz D. R., Landry J. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res. 56:1996;273-279.
51. Mehlen P., Briolay J., Smith L., Diaz-Latoud C., Pauli D., Arrigo A.-P. Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of theDrosophila. Eur. J. Biochem. 215:1993;277-284.
52. Mehlen P., Préville X., Chareyron P., Briolay J., Klemenz R., Arrigo A.-P. Constitutive expression of human hsp27,Drosophila. J. Immunol. 154:1995;363-374.
53. Mehlen P., Préville X., Kretz-Remy C., Arrigo A.-P. Human hsp27,Drosophila. EMBO J. 15:1996;2695-2706.
54. Park Y. M., Han M. Y., Blackburn R. V., Lee Y. J. Overexpression of HSP25 reduces the level of TNF alpha-induced oxidative DNA damage biomarker, 8-hydroxy-2′-deoxyguanosine, in L929 cells. J. Cell. Physiol. 174:1998;27-34.
55. Jäättelä M., Wissing D., Bauer P., Li G. Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J. 11:1992;3507-3512.
56. Jäättelä M. Overexpression of major heat shock protein hsp70 inhibits tumor necrosis factor-induced activation of phospholipase A2. J. Immunol. 151:1993;4286-4294.
57. Arata S., Hamaguchi S., Nose K. Effects of the overexpression of the small heat shock protein, Hsp27, on the sensitivity of human fibroblast cells exposed to oxidative stress. J. Cell. Physiol. 163:1995;458-465.
58. Kretz-Remy C., Mehlen P., Mirault M. E., Arrigo A.-P. Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression. J. Cell Biol. 133:1996;1083-1093.
59. Mirault M. E., Tremblay A., Beaudoin N., Tremblay M. Overexpression of seleno-glutathione peroxidase by gene transfer enhances the resistance of T47D human breast cells to clastogenic oxidants. J. Biol. Chem. 266:1991;20752-20760.
60. Chen E. Y., Cheng A., Lee A., Kuang W. J., Hillier L., Green P., Schlessinger D., Ciccodicola A., D'Urso M. Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome. Genomics. 10:1991;792-800.
61. Hodgson C. P., Solaiman F. Virosomes: Cationic liposomes enhance retroviral transduction. Nat. Biotechnol. 14:1996;339-342.
62. Petit P. X., Lecoeur H., Zorn E., Dauguet C., Mignotte B., Gougeon M. L. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130:1995;157-167.
63. Trounce I., Kim Y., Jun A., Wallace D. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 264:1996;484-509.
64. Préville X., Mehlen P., Fabre-Jonca N., Chaufour S., Kretz-Remy C., Michel M. R., Arrigo A.-P. Biochemical and immunofluorescence analysis of the constitutively expressed hsp27 stress protein in monkey CV-1 cells. J. Biosci. 21:1996;1-14.
65. Arrigo A.-P., Pauli D. Characterization of HSP 27 and three immunologically related proteins duringDrosophila. Exp. Cell Res. 175:1988;169-183.
66. Levine R. L., Garland D., Oliver C. N., Amici A., Climent I., Lenz A. G., Ahn B. W., Shaltiel S., Stadtman E. R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186:1990;464-478.
67. Levine R. L., Williams J. A., Stadtman E. R., Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 233:1994;346-357.
68. 22. Arch. Biochem. Biophys. 292:1992;221-227.
69. Srere P. A. Citrate synthase. Methods Enzymol. 13:1969;3-11.
70. Battistuzzi G., D'Urso M., Toniolo D., Persico G. M., Luzzatto L. Tissue-specific levels of human glucose-6-phosphate dehydrogenase correlate with methylation of specific sites at the 3′ end of the gene. Proc. Natl. Acad. Sci. USA. 82:1985;1465-1469.
71. Tian W. N., Pignatare J. N., Stanton R. C. Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells. J. Biol. Chem. 269:1994;14798-14805.
72. Town M., Athanasiou-Metaxa M., Luzzatto L. Intragenic interspecific complementation of glucose 6-phosphate dehydrogenase in human-hamster cell hybrids. Somatic Cell Mol. Genet. 16:1990;97-108.
73. Habig W. H., Jakoby W. B. Assays for differentiation of glutathioneS. Methods Enzymol. 77:1981;398-405.
74. Aebi H. Catalasein vitro. Methods Enzymol. 105:1984;121-126.
75. Carlberg I., Mannervik B. Glutathione reductase. Methods Enzymol. 113:1985;484-490.
76. Mannervik B. Glutathione peroxidase. Methods Enzymol. 113:1985;490-495.
77. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:1976;248-254.
78. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:1987;156-159.
79. Sambrook, J. Maniatis, T. Frish, E. F. 1989, Molecular Cloning: A Laboratory Manual, J. SambrookT. ManiatisE. F. Frish, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
80. Martini G., Toniolo D., Vulliamy T., Luzzatto L., Dono R., Viglietto G., Paonessa G., D'Urso M., Persico M. G. Structural analysis of the X-linked gene encoding human glucose 6-phosphate dehydrogenase. EMBO J. 5:1986;1849-1855.
81. Grimaldi G., Di Fiore P., Locatelli E. K., Falco J., Blasi F. Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells. EMBO J. 5:1986;855-861.
82. Benatti U., Morelli A., Meloni T., Sparatore B., Salamino F., Michetti M., Melloni E., Pontremoli S., de Flora A. Comparative patterns of 'in vitro' oxidative hemolysis of normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. FEBS Lett. 128:1981;225-229.
83. 22. Cell Stress Chaperones. 3:1998;177-187.
84. Bellomo G., Mirabelli F., Vairetti M., Malormi W. Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: Biochemical and immunocytochemical features. J. Cell. Physiol. 143:1990;118-126.
85. Van den Dobbelsteen D. J., Nobel C. S. I., Schlegel J., Cotgreave I. A., Orrenius S., Slater A. F. G. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J. Biol. Chem. 271:1996;15420-15427.
86. Neuschwander-Tetri B., Roll F. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct. Anal. Biochem. 179:1989;236-241.
87. Agarwal S., Sohal R. S. Aging and protein oxidative damage. Mech. Ageing Dev. 75:1994;11-19.
88. Deas O., Dumont C., Mollereau B., Metivier D., Pasquier C., Bernard-Pomier G., Hirsch F., Charpentier B., Senik A. Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells. Int. Immunol. 9:1997;117-125.
89. Minhas H. S., Thornalley P. J. Comparison of the delivery of reduced glutathione into P388D1 cells by reduced glutathione and its mono- and diethyl ester derivatives. Biochem. Pharmacol. 49:1995;1475-1482.
90. 2. J. Biol. Chem. 263:1988;4704-4711.
91. Hook D., Harding J. Alpha-crystallin acting as a molecular chaperone protects catalase against steroid-induced inactivation. FEBS Lett. 382:1996;281-284.
92. Ganea E., Harding J. J. Molecular chaperones protect against glycation-induced inactivation of glucose-6-phosphate dehydrogenase. Eur. J. Biochem. 231:1995;181-187.
93. Naylor C. E., Rowland P., Basak A. K., Gover S., Mason P. J., Bautista J. M., Vulliamy T. J., Luzzatto L., Adams M. J. Glucose-6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme. Blood. 87:1996;2974-2982.
94. Friguet B., Stadtman E. R., Szweda L. I. Modification of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. Formation of cross-linked protein that inhibits the multicatalytic protease. J. Biol. Chem. 269:1994;21639-21643.
95. Friguet B., Szweda L. I., Stadtman E. R. Susceptibility of glucose-6-phosphate dehydrogenase modified by 4-hydroxy-2-nonenal and metal-catalyzed oxidation to proteolysis by the multicatalytic protease. Arch. Biochem. Biophys. 311:1994;168-173.
96. Babbs C., Steiner M. Detection and quantitation of hydroxyl radical using dimethyl sulfoxide as molecular probe. Methods Enzymol. 186:1990;137-147.
97. Black S. M., Beggs J. D., Hayes J. D., Bartoszek A., Muramatsu M., Sakai M., Wolf C. R. Expression of human glutathioneSSaccharomyces cerevisiae. Biochem. J. 268:1990;309-315.
98. Chao C. C., Huang Y. T., Ma C. M., Chou W. Y., Lin-Chao S. Overexpression of glutathioneS. Mol. Pharmacol. 41:1992;69-75.
99. Hao X. Y., Bergh J., Brodin O., Hellman U., Mannervik B. Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes. Carcinogenesis. 15:1994;1167-1173.
100. Lee F. Y., Siemann D. W. Isolation by flow cytometry of a human ovarian tumor cell subpopulation exhibiting a high glutathione content phenotype and increased resistance to adriamycin. Int. J. Radiat. Oncol. Biol. Phys. 16:1989;1315-1319.
101. Rowley D. A., Halliwell B. DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin. Biochim. Biophys. Acta. 761:1983;86-93.
102. Ehrnsperger M., Graber S., Gaestel M., Buchner J. Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J. 16:1997;221-229.
103. Lee G. J., Roseman A. M., Saibil H. R., Vierling E. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J. 16:1997;659-671.
104. Harding J. J. Post-translational modification of lens proteins in cataract. Lens Eye Toxicol. Res. 8:1991;245-250.
105. Nover L., Scharf K.-D., Neumann D. Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs. Mol. Cell Biol. 9:1989;1298-1308.