Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Feb 2;132(4):577–584. doi: 10.1083/jcb.132.4.577

Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6- phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting

PMCID: PMC2199859  PMID: 8647889

Abstract

We have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS- PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of approximately 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys34 to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys30 to Ala mutation had no biologic consequences, showing the importance of Cys34. Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys30 and Cys34 and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys30 and Cys34 leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys34 is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. 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]
  2. Crise B., Rose J. K. Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor. J Biol Chem. 1992 Jul 5;267(19):13593–13597. [PubMed] [Google Scholar]
  3. Faust P. L., Wall D. A., Perara E., Lingappa V. R., Kornfeld S. Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting. J Cell Biol. 1987 Nov;105(5):1937–1945. doi: 10.1083/jcb.105.5.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gabel C. A., Goldberg D. E., Kornfeld S. Identification and characterization of cells deficient in the mannose 6-phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targeting. Proc Natl Acad Sci U S A. 1983 Feb;80(3):775–779. doi: 10.1073/pnas.80.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Green S. A., Zimmer K. P., Griffiths G., Mellman I. Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. J Cell Biol. 1987 Sep;105(3):1227–1240. doi: 10.1083/jcb.105.3.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Hille-Rehfeld A. Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes. Biochim Biophys Acta. 1995 Jul 17;1241(2):177–194. doi: 10.1016/0304-4157(95)00004-b. [DOI] [PubMed] [Google Scholar]
  8. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  9. Jing S. Q., Trowbridge I. S. Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J. 1987 Feb;6(2):327–331. doi: 10.1002/j.1460-2075.1987.tb04758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Johnson K. F., Chan W., Kornfeld S. Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10010–10014. doi: 10.1073/pnas.87.24.10010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson K. F., Kornfeld S. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J Biol Chem. 1992 Aug 25;267(24):17110–17115. [PubMed] [Google Scholar]
  13. Karnik S. S., Ridge K. D., Bhattacharya S., Khorana H. G. Palmitoylation of bovine opsin and its cysteine mutants in COS cells. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):40–44. doi: 10.1073/pnas.90.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaufman J. F., Krangel M. S., Strominger J. L. Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds. J Biol Chem. 1984 Jun 10;259(11):7230–7238. [PubMed] [Google Scholar]
  15. Kawate N., Menon K. M. Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor. J Biol Chem. 1994 Dec 2;269(48):30651–30658. [PubMed] [Google Scholar]
  16. Koch N., Hämmerling G. J. The HLA-D-associated invariant chain binds palmitic acid at the cysteine adjacent to the membrane segment. J Biol Chem. 1986 Mar 5;261(7):3434–3440. [PubMed] [Google Scholar]
  17. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  18. Liu Y., Fisher D. A., Storm D. R. Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis. Biochemistry. 1993 Oct 12;32(40):10714–10719. doi: 10.1021/bi00091a023. [DOI] [PubMed] [Google Scholar]
  19. Ludwig T., Le Borgne R., Hoflack B. Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly. Trends Cell Biol. 1995 May;5(5):202–206. doi: 10.1016/s0962-8924(00)89000-5. [DOI] [PubMed] [Google Scholar]
  20. Ma Z. M., Grubb J. H., Sly W. S. Cloning, sequencing, and functional characterization of the murine 46-kDa mannose 6-phosphate receptor. J Biol Chem. 1991 Jun 5;266(16):10589–10595. [PubMed] [Google Scholar]
  21. 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]
  22. Messner D. J. The mannose receptor and the cation-dependent form of mannose 6-phosphate receptor have overlapping cellular and subcellular distributions in liver. Arch Biochem Biophys. 1993 Nov 1;306(2):391–401. doi: 10.1006/abbi.1993.1528. [DOI] [PubMed] [Google Scholar]
  23. O'Brien P. J., St Jules R. S., Reddy T. S., Bazan N. G., Zatz M. Acylation of disc membrane rhodopsin may be nonenzymatic. J Biol Chem. 1987 Apr 15;262(11):5210–5215. [PubMed] [Google Scholar]
  24. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  25. Omary M. B., Trowbridge I. S. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed] [Google Scholar]
  26. Ovchinnikov YuA, Abdulaev N. G., Bogachuk A. S. Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated. FEBS Lett. 1988 Mar 28;230(1-2):1–5. doi: 10.1016/0014-5793(88)80628-8. [DOI] [PubMed] [Google Scholar]
  27. Papac D. I., Thornburg K. R., Büllesbach E. E., Crouch R. K., Knapp D. R. Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry. J Biol Chem. 1992 Aug 25;267(24):16889–16894. [PubMed] [Google Scholar]
  28. Rohrer J., Schweizer A., Johnson K. F., Kornfeld S. A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes. J Cell Biol. 1995 Sep;130(6):1297–1306. doi: 10.1083/jcb.130.6.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schlesinger M. J., Magee A. I., Schmidt M. F. Fatty acid acylation of proteins in cultured cells. J Biol Chem. 1980 Nov 10;255(21):10021–10024. [PubMed] [Google Scholar]
  31. Schweizer A., Rohrer J., Kornfeld S. Determination of the structural requirements for palmitoylation of p63. J Biol Chem. 1995 Apr 21;270(16):9638–9644. doi: 10.1074/jbc.270.16.9638. [DOI] [PubMed] [Google Scholar]
  32. Sefton B. M., Buss J. E. The covalent modification of eukaryotic proteins with lipid. J Cell Biol. 1987 Jun;104(6):1449–1453. doi: 10.1083/jcb.104.6.1449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Staufenbiel M. Ankyrin-bound fatty acid turns over rapidly at the erythrocyte plasma membrane. Mol Cell Biol. 1987 Aug;7(8):2981–2984. doi: 10.1128/mcb.7.8.2981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stein M., Meyer H. E., Hasilik A., von Figura K. 46-kDa mannose 6-phosphate-specific receptor: purification, subunit composition, chemical modification. Biol Chem Hoppe Seyler. 1987 Aug;368(8):927–936. doi: 10.1515/bchm3.1987.368.2.927. [DOI] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Veit M., Kretzschmar E., Kuroda K., Garten W., Schmidt M. F., Klenk H. D., Rott R. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. J Virol. 1991 May;65(5):2491–2500. doi: 10.1128/jvi.65.5.2491-2500.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wedegaertner P. B., Chu D. H., Wilson P. T., Levis M. J., Bourne H. R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. J Biol Chem. 1993 Nov 25;268(33):25001–25008. [PubMed] [Google Scholar]
  39. Westcott K. R., Rome L. H. Cation-independent mannose 6-phosphate receptor contains covalently bound fatty acid. J Cell Biochem. 1988 Sep;38(1):23–33. doi: 10.1002/jcb.240380104. [DOI] [PubMed] [Google Scholar]
  40. Zuber M. X., Strittmatter S. M., Fishman M. C. A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature. 1989 Sep 28;341(6240):345–348. doi: 10.1038/341345a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES