A receptor for the selective uptake and degradation of proteins by lysosomes

Science

Volumn 273, Issue 5274, 1996, Pages 501-503

  Cuervo, Ana Maria ^a   Dice, J Fred ^a  

^a TUFTS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; MEMBRANE PROTEIN; RECEPTOR; RIBONUCLEASE A;

ANIMAL CELL; ANIMAL TISSUE; ARTICLE; CHO CELL; HUMAN; LIVER LYSOSOME; LYSOSOME MEMBRANE; NONHUMAN; POLYACRYLAMIDE GEL ELECTROPHORESIS; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN DEGRADATION; PROTEIN EXPRESSION; PROTEIN TRANSPORT; RAT;

EID: 0029837453     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5274.501     Document Type: Article

References (21)

1. W. A. Dunn Jr., Trends Cell Biol. 4, 139 (1994); S. A. Hayes and J. F. Dice, J. Cell Biol. 132, 255 (1996).
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6. F. Aniento, E. Roche, A. M. Cuervo, E. Knecht, ibid., p. 10463; A. M. Cuervo, E. Knecht, S. R. Terlecky, J. F. Dice, Am. J. Physiol. 269, C1200 (1995).
7. F. Aniento, E. Roche, A. M. Cuervo, E. Knecht, ibid., p. 10463; A. M. Cuervo, E. Knecht, S. R. Terlecky, J. F. Dice, Am. J. Physiol. 269, C1200 (1995).
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10. A. M. Cuervo and J. F. Dice, unpublished results.
11. Y. Noguchu et al., Biochem. Biophys. Res. Commun. 164, 1113 (1989).
12. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
13. Not all cultured cells express this selective lysosomal pathway of proteolysis. For example, skin fibroblasts show less of this activity than do lung fibroblasts, and certain transformed cell lines (such as COS cells) have no detectable activity (L. Terlecky, S. R. Terlecky, J. F. Dice, unpublished results).
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15. CHO cells were transfected with human LGP96 cDNA in the plasmid designated pCR-3 (Invitrogen, San Diego, CA) and selected for stable transfectants by resistance to Geneticin.
16. Hybridoma H4B4 was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, IA, under contract NO1-HD-6-2915 from the National Institute of Child Health and Human Development.
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19. 2, 1 mM EDTA, and 0.3% Tween 20] for 12 hours at 4°C. Bound protein was detected by ECL immunoblotting (Amersham International, Buckinghamshire, UK) with a specific antibody to RNase A (4, 5) or by direct exposure to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). In other studies, rat liver lysosomal membrane proteins (2 mg) were solubilized in 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100 and then centrifuged at 100,000g for 30 min. The supernatant was incubated with GAPDH immobilized in a 2-ml Aminolink Plus gel column (Pierce, Rockford, IL) for 2 hours at 25°C. Flow through the column was collected, and after extensive washing, GAPDH-bound proteins were eluted with 10 ml of acetate buffer (pH 4.0) [25 mM sodium acetate (pH 4.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.3% Tween 20] followed by 10 ml of 1 M NaCl in 0.1 M sodium phosphate buffer (pH 7.2).
20. 2 or both were added. Proteolysis was expressed as the percentage of the initial acid-precipitable radio-activity converted to acid-soluble radioactivity.
21. We thank M. Berne for the peptide sequencing. The polyclonal antibody to cathepsin D was a gift of G. Sahagian, Department of Physiology, Tufts University, Boston, MA. This research was funded by a Fundacion Ramon Areces (Spain) and American Liver Foundation postdoctoral fellowships and by NIH grant AG06116.