Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Jan 1;116(1):135–146. doi: 10.1083/jcb.116.1.135

Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein

PMCID: PMC2289273  PMID: 1730740

Abstract

Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Argon Y., Milstein C. Intracellular processing of membrane and secreted immunoglobulin delta-chains. J Immunol. 1984 Sep;133(3):1627–1634. [PubMed] [Google Scholar]
  2. Balch W. E., Rothman J. E. Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys. 1985 Jul;240(1):413–425. doi: 10.1016/0003-9861(85)90046-3. [DOI] [PubMed] [Google Scholar]
  3. Balch W. E., Wagner K. R., Keller D. S. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol. 1987 Mar;104(3):749–760. doi: 10.1083/jcb.104.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beckers C. J., Balch W. E. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol. 1989 Apr;108(4):1245–1256. doi: 10.1083/jcb.108.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonatti S., Migliaccio G., Simons K. Palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum. J Biol Chem. 1989 Jul 25;264(21):12590–12595. [PubMed] [Google Scholar]
  6. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  7. Bretz R., Stäubli W. Detergent influence on rat-liver galactosyltransferase activities towards different acceptors. Eur J Biochem. 1977 Jul 1;77(1):181–192. doi: 10.1111/j.1432-1033.1977.tb11656.x. [DOI] [PubMed] [Google Scholar]
  8. Buell D. N., Fahey J. L. Limited periods of gene expression in immunoglobulin-synthesizing cells. Science. 1969 Jun 27;164(3887):1524–1525. doi: 10.1126/science.164.3887.1524. [DOI] [PubMed] [Google Scholar]
  9. Burke B., Griffiths G., Reggio H., Louvard D., Warren G. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1982;1(12):1621–1628. doi: 10.1002/j.1460-2075.1982.tb01364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burkhardt J. K., Argon Y. Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing. J Cell Sci. 1989 Apr;92(Pt 4):633–642. doi: 10.1242/jcs.92.4.633. [DOI] [PubMed] [Google Scholar]
  11. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  12. Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donaldson J. G., Lippincott-Schwartz J., Bloom G. S., Kreis T. E., Klausner R. D. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. 1990 Dec;111(6 Pt 1):2295–2306. doi: 10.1083/jcb.111.6.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donaldson J. G., Lippincott-Schwartz J., Klausner R. D. Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus. J Cell Biol. 1991 Feb;112(4):579–588. doi: 10.1083/jcb.112.4.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Featherstone C., Griffiths G., Warren G. Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J Cell Biol. 1985 Dec;101(6):2036–2046. doi: 10.1083/jcb.101.6.2036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fries E., Rothman J. E. Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3870–3874. doi: 10.1073/pnas.77.7.3870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glick B. S., Rothman J. E. Possible role for fatty acyl-coenzyme A in intracellular protein transport. Nature. 1987 Mar 19;326(6110):309–312. doi: 10.1038/326309a0. [DOI] [PubMed] [Google Scholar]
  18. Gottlieb C., Skinner A. M., Kornfeld S. Isolation of a clone of Chinese hamster ovary cells deficient in plant lectin-binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1078–1082. doi: 10.1073/pnas.71.4.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Green J., Griffiths G., Louvard D., Quinn P., Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. doi: 10.1016/0022-2836(81)90122-4. [DOI] [PubMed] [Google Scholar]
  20. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  22. Hiller G., Weber K. Golgi detection in mitotic and interphase cells by antibodies to secreted galactosyltransferase. Exp Cell Res. 1982 Nov;142(1):85–94. doi: 10.1016/0014-4827(82)90412-8. [DOI] [PubMed] [Google Scholar]
  23. James G., Olson E. N. Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation. J Biol Chem. 1989 Dec 15;264(35):20998–21006. [PubMed] [Google Scholar]
  24. Jing S. Q., Trowbridge I. S. Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake. J Biol Chem. 1990 Jul 15;265(20):11555–11559. [PubMed] [Google Scholar]
  25. Kabcenell A. K., Atkinson P. H. Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11. J Cell Biol. 1985 Oct;101(4):1270–1280. doi: 10.1083/jcb.101.4.1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klevecz R. R. Automated cell cycle analysis. Methods Cell Biol. 1975;10:157–172. doi: 10.1016/s0091-679x(08)60735-9. [DOI] [PubMed] [Google Scholar]
  27. Kobayashi T., Pagano R. E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface. J Biol Chem. 1989 Apr 5;264(10):5966–5973. [PubMed] [Google Scholar]
  28. Lewis M. J., Pelham H. R. A human homologue of the yeast HDEL receptor. Nature. 1990 Nov 8;348(6297):162–163. doi: 10.1038/348162a0. [DOI] [PubMed] [Google Scholar]
  29. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  30. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lucocq J. M., Berger E. G., Warren G. Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J Cell Biol. 1989 Aug;109(2):463–474. doi: 10.1083/jcb.109.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lucocq J. M., Pryde J. G., Berger E. G., Warren G. A mitotic form of the Golgi apparatus in HeLa cells. J Cell Biol. 1987 Apr;104(4):865–874. doi: 10.1083/jcb.104.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Magee A. I., Courtneidge S. A. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 1985 May;4(5):1137–1144. doi: 10.1002/j.1460-2075.1985.tb03751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Magee A. I., Gutierrez L., McKay I. A., Marshall C. J., Hall A. Dynamic fatty acylation of p21N-ras. EMBO J. 1987 Nov;6(11):3353–3357. doi: 10.1002/j.1460-2075.1987.tb02656.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McIlhinney R. A., Pelly S. J., Chadwick J. K., Cowley G. P. Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways. EMBO J. 1985 May;4(5):1145–1152. doi: 10.1002/j.1460-2075.1985.tb03752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McIlhinney R. A. The fats of life: the importance and function of protein acylation. Trends Biochem Sci. 1990 Oct;15(10):387–391. doi: 10.1016/0968-0004(90)90237-6. [DOI] [PubMed] [Google Scholar]
  38. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  39. Misumi Y., Misumi Y., Miki K., Takatsuki A., Tamura G., Ikehara Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem. 1986 Aug 25;261(24):11398–11403. [PubMed] [Google Scholar]
  40. Olson E. N. Modification of proteins with covalent lipids. Prog Lipid Res. 1988;27(3):177–197. doi: 10.1016/0163-7827(88)90012-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Olson E. N., Spizz G. Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation. J Biol Chem. 1986 Feb 15;261(5):2458–2466. [PubMed] [Google Scholar]
  42. Omary M. B., Trowbridge I. S. Covalent binding of fatty acid to the transferrin receptor in cultured human cells. J Biol Chem. 1981 May 25;256(10):4715–4718. [PubMed] [Google Scholar]
  43. Orci L., Malhotra V., Amherdt M., Serafini T., Rothman J. E. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell. 1989 Feb 10;56(3):357–368. doi: 10.1016/0092-8674(89)90239-0. [DOI] [PubMed] [Google Scholar]
  44. Orci L., Tagaya M., Amherdt M., Perrelet A., Donaldson J. G., Lippincott-Schwartz J., Klausner R. D., Rothman J. E. Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell. 1991 Mar 22;64(6):1183–1195. doi: 10.1016/0092-8674(91)90273-2. [DOI] [PubMed] [Google Scholar]
  45. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Percy A. K., Moore J. F., Plishker G. A., Waymire J. C. Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells. Neurochem Res. 1991 Apr;16(4):505–511. doi: 10.1007/BF00965573. [DOI] [PubMed] [Google Scholar]
  48. Pfanner N., Glick B. S., Arden S. R., Rothman J. E. Fatty acylation promotes fusion of transport vesicles with Golgi cisternae. J Cell Biol. 1990 Apr;110(4):955–961. doi: 10.1083/jcb.110.4.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pfanner N., Orci L., Glick B. S., Amherdt M., Arden S. R., Malhotra V., Rothman J. E. Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae. Cell. 1989 Oct 6;59(1):95–102. doi: 10.1016/0092-8674(89)90872-6. [DOI] [PubMed] [Google Scholar]
  50. Rothman J. E., Orci L. Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport. FASEB J. 1990 Mar;4(5):1460–1468. doi: 10.1096/fasebj.4.5.2407590. [DOI] [PubMed] [Google Scholar]
  51. Saraste J., Palade G. E., Farquhar M. G. Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein. J Cell Biol. 1987 Nov;105(5):2021–2029. doi: 10.1083/jcb.105.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Skene J. H., Virág I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol. 1989 Feb;108(2):613–624. doi: 10.1083/jcb.108.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Staufenbiel M. Fatty acids covalently bound to erythrocyte proteins undergo a differential turnover in vivo. J Biol Chem. 1988 Sep 25;263(27):13615–13622. [PubMed] [Google Scholar]
  54. Storrie B., Madden E. A. Isolation of subcellular organelles. Methods Enzymol. 1990;182:203–225. doi: 10.1016/0076-6879(90)82018-w. [DOI] [PubMed] [Google Scholar]
  55. Tartakoff A. M. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell. 1983 Apr;32(4):1026–1028. doi: 10.1016/0092-8674(83)90286-6. [DOI] [PubMed] [Google Scholar]
  56. Warren G. Intracellular transport. Vesicular consumption. Nature. 1990 May 31;345(6274):382–383. doi: 10.1038/345382a0. [DOI] [PubMed] [Google Scholar]
  57. Waters M. G., Serafini T., Rothman J. E. 'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature. 1991 Jan 17;349(6306):248–251. doi: 10.1038/349248a0. [DOI] [PubMed] [Google Scholar]
  58. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  59. Wilcox C. A., Olson E. N. The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane. Biochemistry. 1987 Feb 24;26(4):1029–1036. doi: 10.1021/bi00378a008. [DOI] [PubMed] [Google Scholar]
  60. Woodman P. G., Warren G. Fusion between vesicles from the pathway of receptor-mediated endocytosis in a cell-free system. Eur J Biochem. 1988 Apr 5;173(1):101–108. doi: 10.1111/j.1432-1033.1988.tb13972.x. [DOI] [PubMed] [Google Scholar]
  61. Zeligs J. D., Wollman S. H. Mitosis in rat thyroid epithelial cells in vivo. I. Ultrastructural changes in cytoplasmic organelles during the mitotic cycle. J Ultrastruct Res. 1979 Jan;66(1):53–77. doi: 10.1016/s0022-5320(79)80065-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES