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. 1992 Apr 2;117(2):311–325. doi: 10.1083/jcb.117.2.311

Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane

PMCID: PMC2289424  PMID: 1560028

Abstract

We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail.

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Selected References

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  1. 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]
  2. 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]
  3. Cha Y., Holland S. M., August J. T. The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins. J Biol Chem. 1990 Mar 25;265(9):5008–5013. [PubMed] [Google Scholar]
  4. Chen J. W., Cha Y., Yuksel K. U., Gracy R. W., August J. T. Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens. J Biol Chem. 1988 Jun 25;263(18):8754–8758. [PubMed] [Google Scholar]
  5. 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]
  6. Davis C. G., Lehrman M. A., Russell D. W., Anderson R. G., Brown M. S., Goldstein J. L. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell. 1986 Apr 11;45(1):15–24. doi: 10.1016/0092-8674(86)90533-7. [DOI] [PubMed] [Google Scholar]
  7. Duden R., Griffiths G., Frank R., Argos P., Kreis T. E. Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell. 1991 Feb 8;64(3):649–665. doi: 10.1016/0092-8674(91)90248-w. [DOI] [PubMed] [Google Scholar]
  8. Fambrough D. M., Takeyasu K., Lippincott-Schwarz J., Siegel N. R. Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA. J Cell Biol. 1988 Jan;106(1):61–67. doi: 10.1083/jcb.106.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Furuno K., Ishikawa T., Akasaki K., Yano S., Tanaka Y., Yamaguchi Y., Tsuji H., Himeno M., Kato K. Morphological localization of a major lysosomal membrane glycoprotein in the endocytic membrane system. J Biochem. 1989 Oct;106(4):708–716. doi: 10.1093/oxfordjournals.jbchem.a122921. [DOI] [PubMed] [Google Scholar]
  10. Furuno K., Yano S., Akasaki K., Tanaka Y., Yamaguchi Y., Tsuji H., Himeno M., Kato K. Biochemical analysis of the movement of a major lysosomal membrane glycoprotein in the endocytic membrane system. J Biochem. 1989 Oct;106(4):717–722. doi: 10.1093/oxfordjournals.jbchem.a122922. [DOI] [PubMed] [Google Scholar]
  11. Glickman J. N., Conibear E., Pearse B. M. Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor. EMBO J. 1989 Apr;8(4):1041–1047. doi: 10.1002/j.1460-2075.1989.tb03471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Granger B. L., Green S. A., Gabel C. A., Howe C. L., Mellman I., Helenius A. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells. J Biol Chem. 1990 Jul 15;265(20):12036–12043. [PubMed] [Google Scholar]
  13. 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]
  14. Himeno M., Noguchi Y., Sasaki H., Tanaka Y., Furuno K., Kono A., Sakaki Y., Kato K. Isolation and sequencing of a cDNA clone encoding 107 kDa sialoglycoprotein in rat liver lysosomal membranes. FEBS Lett. 1989 Feb 27;244(2):351–356. doi: 10.1016/0014-5793(89)80561-7. [DOI] [PubMed] [Google Scholar]
  15. Horwich A. L., Fenton W. A., Firgaira F. A., Fox J. E., Kolansky D., Mellman I. S., Rosenberg L. E. Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor. J Cell Biol. 1985 May;100(5):1515–1521. doi: 10.1083/jcb.100.5.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Howe C. L., Granger B. L., Hull M., Green S. A., Gabel C. A., Helenius A., Mellman I. Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7577–7581. doi: 10.1073/pnas.85.20.7577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunziker W., Harter C., Matter K., Mellman I. Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell. 1991 Sep 6;66(5):907–920. doi: 10.1016/0092-8674(91)90437-4. [DOI] [PubMed] [Google Scholar]
  18. Kaufman R. J. Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 1990;185:537–566. doi: 10.1016/0076-6879(90)85044-o. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Ktistakis N. T., Thomas D., Roth M. G. Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins. J Cell Biol. 1990 Oct;111(4):1393–1407. doi: 10.1083/jcb.111.4.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lewis V., Green S. A., Marsh M., Vihko P., Helenius A., Mellman I. Glycoproteins of the lysosomal membrane. J Cell Biol. 1985 Jun;100(6):1839–1847. doi: 10.1083/jcb.100.6.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lippincott-Schwartz J., Fambrough D. M. Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein. J Cell Biol. 1986 May;102(5):1593–1605. doi: 10.1083/jcb.102.5.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Mane S. M., Marzella L., Bainton D. F., Holt V. K., Cha Y., Hildreth J. E., August J. T. Purification and characterization of human lysosomal membrane glycoproteins. Arch Biochem Biophys. 1989 Jan;268(1):360–378. doi: 10.1016/0003-9861(89)90597-3. [DOI] [PubMed] [Google Scholar]
  26. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  27. Miettinen H. M., Rose J. K., Mellman I. Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity. Cell. 1989 Jul 28;58(2):317–327. doi: 10.1016/0092-8674(89)90846-5. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Noguchi Y., Himeno M., Sasaki H., Tanaka Y., Kono A., Sakaki Y., Kato K. Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes. Biochem Biophys Res Commun. 1989 Nov 15;164(3):1113–1120. doi: 10.1016/0006-291x(89)91784-1. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Serafini T., Stenbeck G., Brecht A., Lottspeich F., Orci L., Rothman J. E., Wieland F. T. A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin. Nature. 1991 Jan 17;349(6306):215–220. doi: 10.1038/349215a0. [DOI] [PubMed] [Google Scholar]
  34. Stevens T. H., Rothman J. H., Payne G. S., Schekman R. Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol. 1986 May;102(5):1551–1557. doi: 10.1083/jcb.102.5.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Viitala J., Carlsson S. R., Siebert P. D., Fukuda M. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent Mr approximately equal to 120,000. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3743–3747. doi: 10.1073/pnas.85.11.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]

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