Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Feb 2;132(4):565–576. doi: 10.1083/jcb.132.4.565

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane

PMCID: PMC2199866  PMID: 8647888

Abstract

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Aroeti B., Mostov K. E. Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids. EMBO J. 1994 May 15;13(10):2297–2304. doi: 10.1002/j.1460-2075.1994.tb06513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bos K., Wraight C., Stanley K. K. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 1993 May;12(5):2219–2228. doi: 10.1002/j.1460-2075.1993.tb05870.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braun M., Waheed A., von Figura K. Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 1989 Dec 1;8(12):3633–3640. doi: 10.1002/j.1460-2075.1989.tb08537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bretscher M. S., Lutter R. A new method for detecting endocytosed proteins. EMBO J. 1988 Dec 20;7(13):4087–4092. doi: 10.1002/j.1460-2075.1988.tb03302.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Canfield W. M., Johnson K. F., Ye R. D., Gregory W., Kornfeld S. Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail. J Biol Chem. 1991 Mar 25;266(9):5682–5688. [PubMed] [Google Scholar]
  6. Carlsson S. R., Fukuda M. The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways. Arch Biochem Biophys. 1992 Aug 1;296(2):630–639. doi: 10.1016/0003-9861(92)90619-8. [DOI] [PubMed] [Google Scholar]
  7. Carlsson S. R., Roth J., Piller F., Fukuda M. Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem. 1988 Dec 15;263(35):18911–18919. [PubMed] [Google Scholar]
  8. Chen J. W., Chen G. L., D'Souza M. P., Murphy T. L., August J. T. Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2. Biochem Soc Symp. 1986;51:97–112. [PubMed] [Google Scholar]
  9. D'Souza M. P., August J. T. A kinetic analysis of biosynthesis and localization of a lysosome-associated membrane glycoprotein. Arch Biochem Biophys. 1986 Sep;249(2):522–532. doi: 10.1016/0003-9861(86)90030-5. [DOI] [PubMed] [Google Scholar]
  10. Eberle W., Sander C., Klaus W., Schmidt B., von Figura K., Peters C. The essential tyrosine of the internalization signal in lysosomal acid phosphatase is part of a beta turn. Cell. 1991 Dec 20;67(6):1203–1209. doi: 10.1016/0092-8674(91)90296-b. [DOI] [PubMed] [Google Scholar]
  11. Fuhlbrigge R. C., Fine S. M., Unanue E. R., Chaplin D. D. Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5649–5653. doi: 10.1073/pnas.85.15.5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukuda M. Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem. 1991 Nov 15;266(32):21327–21330. [PubMed] [Google Scholar]
  13. 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]
  14. Green S. A., Setiadi H., McEver R. P., Kelly R. B. The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J Cell Biol. 1994 Feb;124(4):435–448. doi: 10.1083/jcb.124.4.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  17. Guarnieri F. G., Arterburn L. M., Penno M. B., Cha Y., August J. T. The motif Tyr-X-X-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1. J Biol Chem. 1993 Jan 25;268(3):1941–1946. [PubMed] [Google Scholar]
  18. Harter C., Mellman I. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol. 1992 Apr;117(2):311–325. doi: 10.1083/jcb.117.2.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Humphrey J. S., Peters P. J., Yuan L. C., Bonifacino J. S. Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence. J Cell Biol. 1993 Mar;120(5):1123–1135. doi: 10.1083/jcb.120.5.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Höning S., Hunziker W. Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells. J Cell Biol. 1995 Feb;128(3):321–332. doi: 10.1083/jcb.128.3.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jadot M., Canfield W. M., Gregory W., Kornfeld S. Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. J Biol Chem. 1992 Jun 5;267(16):11069–11077. [PubMed] [Google Scholar]
  22. 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]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lippincott-Schwartz J., Fambrough D. M. Cycling of the integral membrane glycoprotein, LEP100, between plasma membrane and lysosomes: kinetic and morphological analysis. Cell. 1987 Jun 5;49(5):669–677. doi: 10.1016/0092-8674(87)90543-5. [DOI] [PubMed] [Google Scholar]
  25. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  26. Ludwig T., Munier-Lehmann H., Bauer U., Hollinshead M., Ovitt C., Lobel P., Hoflack B. Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts. EMBO J. 1994 Aug 1;13(15):3430–3437. doi: 10.1002/j.1460-2075.1994.tb06648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marks M. S., Roche P. A., van Donselaar E., Woodruff L., Peters P. J., Bonifacino J. S. A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments. J Cell Biol. 1995 Oct;131(2):351–369. doi: 10.1083/jcb.131.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mathews P. M., Martinie J. B., Fambrough D. M. The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes. J Cell Biol. 1992 Sep;118(5):1027–1040. doi: 10.1083/jcb.118.5.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matter K., Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol. 1994 Aug;6(4):545–554. doi: 10.1016/0955-0674(94)90075-2. [DOI] [PubMed] [Google Scholar]
  30. Matter K., Whitney J. A., Yamamoto E. M., Mellman I. Common signals control low density lipoprotein receptor sorting in endosomes and the Golgi complex of MDCK cells. Cell. 1993 Sep 24;74(6):1053–1064. doi: 10.1016/0092-8674(93)90727-8. [DOI] [PubMed] [Google Scholar]
  31. Mayor S., Presley J. F., Maxfield F. R. Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J Cell Biol. 1993 Jun;121(6):1257–1269. doi: 10.1083/jcb.121.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Metzelaar M. J., Wijngaard P. L., Peters P. J., Sixma J. J., Nieuwenhuis H. K., Clevers H. C. CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem. 1991 Feb 15;266(5):3239–3245. [PubMed] [Google Scholar]
  33. Nabi I. R., Le Bivic A., Fambrough D., Rodriguez-Boulan E. An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes. J Cell Biol. 1991 Dec;115(6):1573–1584. doi: 10.1083/jcb.115.6.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ogata S., Fukuda M. Lysosomal targeting of Limp II membrane glycoprotein requires a novel Leu-Ile motif at a particular position in its cytoplasmic tail. J Biol Chem. 1994 Feb 18;269(7):5210–5217. [PubMed] [Google Scholar]
  35. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  36. Peters C., Braun M., Weber B., Wendland M., Schmidt B., Pohlmann R., Waheed A., von Figura K. Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes. EMBO J. 1990 Nov;9(11):3497–3506. doi: 10.1002/j.1460-2075.1990.tb07558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Russell D. G. Immunoelectron microscopy of endosomal trafficking in macrophages infected with microbial pathogens. Methods Cell Biol. 1994;45:277–288. doi: 10.1016/s0091-679x(08)61857-9. [DOI] [PubMed] [Google Scholar]
  39. Russell D. G., Xu S., Chakraborty P. Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages. J Cell Sci. 1992 Dec;103(Pt 4):1193–1210. doi: 10.1242/jcs.103.4.1193. [DOI] [PubMed] [Google Scholar]
  40. Sandoval I. V., Arredondo J. J., Alcalde J., Gonzalez Noriega A., Vandekerckhove J., Jimenez M. A., Rico M. The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes. J Biol Chem. 1994 Mar 4;269(9):6622–6631. [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schweizer A., Fransen J. A., Bächi T., Ginsel L., Hauri H. P. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J Cell Biol. 1988 Nov;107(5):1643–1653. doi: 10.1083/jcb.107.5.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tanaka Y., Yano S., Okada K., Ishikawa T., Himeno M., Kato K. Lysosomal acid phosphatase is transported via endosomes to lysosomes. Biochem Biophys Res Commun. 1990 Feb 14;166(3):1176–1182. doi: 10.1016/0006-291x(90)90990-5. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  46. Vega M. A., Rodriguez F., Seguí B., Calés C., Alcalde J., Sandoval I. V. Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes. J Biol Chem. 1991 Sep 5;266(25):16269–16272. [PubMed] [Google Scholar]
  47. 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]
  48. Williams M. A., Fukuda M. Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail. J Cell Biol. 1990 Sep;111(3):955–966. doi: 10.1083/jcb.111.3.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wong S. H., Hong W. The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization. J Biol Chem. 1993 Oct 25;268(30):22853–22862. [PubMed] [Google Scholar]

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

RESOURCES