Analysis of Protein Transport to Lysosomes

Beat E. Schaub1, Prashant Nair1, Jack Rohrer1

1 University of Zurich, Zurich
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.8
DOI:  10.1002/0471143030.cb1508s27
Online Posting Date:  July, 2005
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Abstract

Lysosomes are terminal degradative organelles that are found in all higher eukaryotic cells. The biogenesis of lysosomes involves the transport of various acid hydrolases and transmembrane glycoproteins from their site of synthesis in the endoplasmic reticulum through the biosynthetic and endocytic pathways. Protein transport to lysosomes can be studied by a combination of techniques based on the separation of intracellular organelles. Percoll density gradient centrifugation has long been the method of choice for separating lysosomes from other organelles in cell homogenates, and accordingly, this unit describes protocols for obtaining reasonably pure lysosomal fractions from mammalian cells using Percoll density gradient separation.

Keywords: cell fractionation; ball‐bearing homogenizer; separation; organelles; density gradient; ultracentrifugation; lysosomes; endosomes; marker proteins; cation‐dependent mannose‐6‐phosphate receptor; Lamp‐1; β‐hexosaminidase; colorimetry

     
 
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Table of Contents

  • Basic Protocol 1: Fractionation of Cells Using a Self‐Forming Percoll Density Gradient
  • Support Protocol 1: β‐Hexosaminidase Assay to Check the Efficiency of Fractionation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fractionation of Cells Using a Self‐Forming Percoll Density Gradient

  Materials
  • Mouse D9 L (Rec) fibroblast cell line (CRL‐2648 mouse L cells available from ATCC; for mutant strain, contact Dr. Stuart Kornfeld at ) or other cell line of choice (e.g., HeLa)
  • Growth medium: e.g., Invitrogen α‐minimal essential medium supplemented with 10% v/v fetal bovine serum, with or without 500 µg/ml geneticin
  • Growth medium containing 100 µM pepstatin A (from 30 mM stock in dimethyl sulfoxide; Sigma) and 100 µM leupeptin (from 30 mM stock in H 2O; Sigma)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10× and 1× homogenization buffer (see recipe)
  • 1× homogenization buffer/17.5% (v/v) Percoll solution (see recipe)
  • 10% (v/v) Triton X‐100
  • 500× protease inhibitor cocktail (see recipe)
  • 3× protein gel sample buffer (see recipe)
  • 10‐cm‐diameter Falcon tissue culture dishes (BD Biosciences)
  • Rubber or plastic cell scraper
  • Refrigerated centrifuge with swinging‐bucket rotor suitable for 15‐ml tubes (e.g., Heraeus Cryofuge M7000)
  • Ball‐bearing homogenizer (clearance, 16 µm; Isobiotec) or 25‐G needle attached to 1‐ml syringe
  • 16 × 76–mm Ultra‐Clear centrifuge tubes with metal screw caps (Beckman Coulter)
  • Refrigerated ultracentrifuge with Beckman Ti50 rotor (or equivalent) and Beckman TLA 100.3 rotor (or equivalent)
  • 20‐G needles
  • 3‐ml thick‐walled polycarbonate ultracentrifuge tubes (Beckman Coulter)
  • 1.5‐ml polyallomer microcentrifuge tubes (Beckman Coulter) with suitable adaptors
  • Adaptors for centrifuging 1.5‐ml microcentrifuge tubes in Beckman TLA 100.3 rotor (or equivalent)
NOTE: After cell culturing, perform all steps on ice, and cool all centrifuge parts to 4°C before use.

Support Protocol 1: β‐Hexosaminidase Assay to Check the Efficiency of Fractionation

  Materials
  • Percoll gradient fractions/pools (see protocol 1)
  • β‐Hexosaminidase substrate mix (see recipe)
  • 0.2 M Na 2CO 3
  • Spectrophotometer capable of reading at 400 nm
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Figures

Videos

Literature Cited

Literature Cited
   Barriocanal, J.G., Bonifacino, J.S., Yuan, L., and Sandoval, I.V. 1986. Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N‐linked carbohydrate‐independent mechanism of three lysosomal integral membrane proteins. J. Biol. Chem. 261:16755‐16763.
   Bonifacino, J.S. and Dell'angelica, E.C. 1999. Molecular bases for the recognition of tyrosine‐based sorting signals. J. Cell. Biol. 145:923‐926.
   Braun, M., Waheed, A., and von Figura, K. 1989. Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 8:3633‐3640.
   D'Souza, M.P. and August, J.T. 1986. A kinetic analysis of biosynthesis and localization of a lysosome‐associated membrane glycoprotein. Arch. Biochem. Biophys. 249:522‐532.
   Gottschalk, S., Waheed, A., Schmidt, B., Laidler, P., and von Figura, K. 1989. Sequential processing of lysosomal acid phosphatase by a cytoplasmic thiol proteinase and a lysosomal aspartyl proteinase. EMBO J. 8:3215‐3219.
   Green, S.A., Zimmer, K.P., Griffiths, G., and Mellman, I. 1987. Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. J. Cell. Biol. 105:1227‐1240.
   Griffiths, G., Hoflack, B., Simons, K., Mellman, I., and Kornfeld, S. 1988. The mannose 6‐phosphate receptor and the biogenesis of lysosomes. Cell 52:329‐341.
   Hunziker, W. and Geuze, H.J. 1996. Intracellular trafficking of lysosomal membrane proteins. Bioessays 18:379‐389.
   Lehmann, L.E., Eberle, W., Krull, S., Prill, V., Schmidt, B., Sander, C., von Figura, K., and Peters, C. 1992. The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV. EMBO J. 11:4391‐4399.
   Luzio, J.P., Poupon, V., Lindsay, M.R., Mullock, B.M., Piper, R.C., and Pryor, P.R. 2003. Membrane dynamics and the biogenesis of lysosomes. Mol. Membr. Biol. 20:141‐154.
   Nair, P., Schaub, B.E., and Rohrer, J. 2003. Characterization of the endosomal sorting signal of the cation‐dependent mannose 6‐phosphate receptor. J. Biol. Chem. 278:24753‐24758.
   Peters, C. and von Figura, K. 1994. Biogenesis of lysosomal membranes. FEBS Lett. 346:108‐114.
   Peters, C., Braun, M., Weber, B., Wendland, M., Schmidt, B., Pohlmann, R., Waheed, A., and von Figura, K. 1990. 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. 9:3497‐3506.
   Rohrer, J., Schweizer, A., Johnson, K.F., and Kornfeld, S. 1995. A determinant in the cytoplasmic tail of the cation‐dependent mannose 6‐ phosphate receptor prevents trafficking to lysosomes. J. Cell. Biol. 130:1297‐1306.
   Rohrer, J., Schweizer, A., Russell, D., and Kornfeld, S. 1996. The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane. J. Cell. Biol. 132:565‐576.
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   Schweizer, A., Kornfeld, S., and Rohrer, J. 1997. Proper sorting of the cation‐dependent mannose 6‐phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail. Proc. Natl. Acad. Sci. U.S.A. 94:14471‐14476.
   Schweizer, A., Stahl, P.D., and Rohrer, J. 2000. A di‐aromatic motif in the cytosolic tail of the mannose receptor mediates endosomal sorting. J. Biol. Chem. 275:29694‐29700.
   Storrie, B. and Desjardins, M. 1996. The biogenesis of lysosomes: Is it a kiss and run, continuous fusion and fission process? Bioessays 18:895‐903.
   Varki, A., Reitman, M.L., Vannier, A., Kornfeld, S., Grubb, J.H., and Sly, W.S. 1982. Demonstration of the heterozygous state for I‐cell disease and pseudo‐Hurler polydystrophy by assay of N‐acetylglucosaminylphosphotransferase in white blood cells and fibroblasts. Am. J. Hum. Genet. 34:717‐729.
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   Williams, M.A. and Fukuda, M. 1990. Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail. J. Cell. Biol. 111:955‐966.
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