Endocytic Organelles: Methods for Preparation and Analysis


Endocytic organelles can be prepared using a variety of techniques. Methods include the homogenisation of cells, the isolation of endosomes by density centrifugation, electromigratory techniques, immunoisolation and fluorescence‐activated organelle sorting (FAOS). The integration of these biochemical techniques with large‐scale proteomics has lead to the rapid expansion of the organelle proteomes characterised. Unfortunately, several limitations have been uncovered. First, the isolation of pure endosomal compartments is rarely achieved. Second, the constant flux of proteins from and to a specific organelle leads to variations in proteome composition that are often time‐ and signalling‐dependent. Innovative approaches such as protein correlation profiling (PCP) and localisation of organelles by isotype tagging (LOPIT) may, in the near future, bring us closer to our aim: the generation of a detailed and reliable organelle proteome chart that accounts for time‐ and signalling‐dependent fluxes as well as variations in the posttranslational modifications (PTMs) observed.

Key Concepts:

  • The quality of subcellular organelle purification depends to a great extent on the homogenisation procedure.

  • Upon homogenisation, several methods can be employed to enrich the organelle of choice (density centrifugation, electromigratory techniques, immunoisolation and FAOS).

  • Subcellular organelle proteomes have been uncovered by the powerful combination of cell biology/biochemistry techniques with large‐scale proteomics.

  • Innovative methodologies are being established to overcome some of the caveats observed on the initial organelle proteome studies.

  • Future organelle proteome studies will focus on the dynamics of protein association with organelles (time and signalling specificity). They will also focus on posttranslational modifications and their direct impact on protein function.

Keywords: subcellular fractionation; endocytosis; endosomes; membrane transport; cellular uptake; organelle proteomics

Figure 1.

Step gradient tube after ultracentrifugation. Organelles appear at distinct interfaces as milky bands. Reproduced from de Araújo and Huber , with permission from Springer.

Figure 2.

Overview of available methods to characterise an organelle proteome. Examples of biochemical and proteomics methods used in organelle proteomics. Single organelle flow analysis (SOFA), (FAOS), (FFE), high resolution gradient electrophoresis (DGE), one‐dimensional gel electrophoresis (1D), (iTRAQ), liquid chromatography tandem mass spectrometry (LC‐MS/MS), fluorescence two‐dimensional difference gel electrophoresis (2D‐DIGE), (MALDI‐MS), protein correlation profiling (PCP) and (LOPIT).



Aebersold R and Mann M (2003) Mass spectrometry‐based proteomics. Nature 422(6928): 198–207.

Ajioka RS and Kaplan J (1987) Characterization of endocytic compartments using the horseradish peroxidase‐diaminobenzidine density shift technique. Journal of Cell Biology 104: 77–85.

de Araújo ME and Huber LA (2007) Subcellular fractionation. Methods in Molecular Biology 357: 73–85.

Bagshaw RD, Mahuran DJ and Callahan JW (2005) A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Molecular and Cellular Proteomics 4(2): 133–143.

Balbis A, Parmar A, Wang Ye, Baquiran G and Posner BI (2007) Compartmentalization of signaling‐competent epidermal growth factor receptors in endosomes. Endocrinology 148(6): 2944–2954.

Blonder J, Goshe MB, Moore RJ et al. (2002) Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography‐tandem mass spectrometry. Journal of Proteome Research 1(4): 351–360.

Boeck G, Steinlein P, Haberfellner M, Gruenberg J and Huber LA (1998) Fluorescence‐activated sorting of endocytic organelles. In: Celis JE (ed.) Cell Biology: A Laboratory Handbook, 2nd edn, pp. 63–69. San Diego: Academic Press.

Boeck G, Steinlein P and Huber LA (1997) Cell biologists sort things out: analysis and purification of intracellular organelles by flow cytometry. Trends in Cell Biology 7: 499–503.

Brandhorst D, Zwilling D, Rizzoli S et al. (2006) Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. Proceedings of the National Academy of Sciences of the USA 103(8): 2701–2706.

Bøyum A, Fjerdingstad HB, Martinsen I, Lea T and Løvhaug D (2002) Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: Monocyte depletion depending upon activation of membrane potassium channels. Scandinavian Journal of Immunology 56: 76–84.

Choudhary C and Mann M (2010) Decoding signalling networks by mass spectrometry‐based proteomics. Nature Reviews. Molecular Cell Biology 11(6): 427–439.

Clague MJ (1998) Molecular aspects of the endocytic pathway. Biochemical Journal 336: 271–282.

Conradt B, Shaw J, Vida T, Emr S and Wickner W (1992) In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. Journal of Cell Biology 119: 1469–1479.

Cossarizza A, Ceccarelli D and Masini A (1996) Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Experimental Cell Research 222(1): 84–94.

Ellinger I, Klapper H, Courtoy PJ, Vaerman JP and Fuchs R (2002) Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: analysis by free‐flow electrophoresis. Electrophoresis 23(13): 2117–2129.

Fialka I, Steinlein P, Ahorn H et al. (1999) Identification of syntenin as a protein of the apical early endocytic compartment in Madin‐Darby canine kidney cells. Journal of Biological Chemistry 274: 26233–26239.

Fischer von Mollard G, Stahl B, Walch‐Solimena C et al. (1994) Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway. European Journal of Cell Biology 65: 319–326.

Fleischer S and Kervina M (1974a) Long‐term preservation of liver for subcellular fractionation. Methods in Enzymology 31: 3–6.

Fleischer S and Kervina M (1974b) Subcellular fractionation of rat liver. Methods in Enzymology 31: 6–41.

Foster LJ, de Hoog CL, Zhang Y et al. (2006) A mammalian organelle map by protein correlation profiling. Cell 125(1): 187–199.

Fuchs R and Ellinger I (2002) Free‐flow electrophoretic analysis of endosome subpopulations of rat hepatocytes. Current Protocols in Cell Biology, Chapter 3:Unit 3.11.

Gagnon E, Duclos S, Rondeau C et al. (2002) Endoplasmic reticulum‐mediated phagocytosis is a mechanism of entry into macrophages. Cell 110(1): 119–131.

Gauthier DJ, Sobota JA, Ferraro F, Mains RE and Lazure C (2008) Flow cytometry‐assisted purification and proteomic analysis of the corticotropes dense‐core secretory granules. Proteomics 8(18): 3848–3861. 10.1002/pmic.200700969.

Gruenberg J and Gorvel JP (1992) In vitro reconstitution of endocytic vesicle fusion. In: Magee AI and Wileman T (eds) Protein Targetting, a Practical Approach, pp. 187–215. Oxford: Oxford University Press.

Gruenberg J, Griffiths G and Howell KE (1989) Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. Journal of Cell Biology 108: 1301–1316.

Gruenberg JE and Howell KE (1986) Reconstitution of vesicle fusions occurring in endocytosis with a cell‐free system. EMBO Journal 5(12): 3091–3101.

Gygi SP, Rist B, Gerber SA et al. (1999) Quantitative analysis of complex protein mixtures using isotope‐coded affinity tags. Nature Biotechnology 17: 994–999.

Henley JR, Krueger EW, Oswald BJ and McNiven MA (1998) Dynamin‐mediated internalization of caveolae. Journal of Cell Biology 141: 85–99.

Houde M, Bertholet S, Gagnon E et al. (2003) Phagosomes are competent organelles for antigen cross‐presentation. Nature 425(6956): 402–406.

Jahraus A, Tjelle TE, Berg T et al. (1998) In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages. Journal of Biological Chemistry 273(46): 30379–30390.

Lilley KS and Friedman DB (2004) All about DIGE: quantification technology for differential‐display 2D‐gel proteomics. Expert Review of Proteomics 1(4): 401–409.

Lindner R (2001) One‐step separation of endocytic organelles, Golgi/trans‐Golgi network and plasma membrane by density gradient electrophoresis. Electrophoresis 22(3): 386–393.

Mears R, Craven RA, Hanrahan S et al. (2004) Proteomic analysis of melanoma‐derived exosomes by two‐dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 4(12): 4019–4031.

Morre DJ, Lawrence J, Safranski K, Hammond T and Morre DM (1994) Experimental basis for separation of membrane vesicles by preparative free‐flow electrophoresis. Journal of Chromatography A 668: 201–213.

Murphy RF (1995) Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid‐phase endocytosis. Proceedings of the National Academy of Sciences of the USA 82: 8523–8526.

Pasquali C, Fialka I and Huber LA (1999) Subcellular fractionation, electromigration analysis and mapping of organelles. Journal of Chromatography B 722: 89–102.

Pisitkun T, Shen RF and Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proceedings of the National Academy of Sciences of the USA 101(36): 13368–13373.

Rajotte D, Stearns CD and Kabcenell AK (2003) Isolation of mast cell secretory lysosomes using flow cytometry. Cytometry 55(2): 94–101.

Rizzoli S, Bethani I, Zwilling D et al. (2006) Evidence for early endosome‐like fusion of recently endocytosed synaptic vesicles. Traffic 7(9): 1163–1176.

Ross PL, Huang YN, Marchese JN et al. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine‐reactive isobaric tagging reagents. Molecular and Cellular Proteomics 3(12): 1154–1169.

Sadowski PG, Dunkley TP, Shadforth IP et al. (2006) Quantitative proteomic approach to study subcellular localization of membrane proteins. Nature Protocols 1(4): 1778–1789.

Sadowski PG, Groen AJ, Dupree P and Lilley KS (2008) Sub‐cellular localization of membrane proteins. Proteomics 8(19): 3991–4011.

Schmid SL, Fuchs R, Male P and Mellman I (1988) Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell 52: 73–83.

Steele‐Mortimer O, Clague MJ, Huber LA et al. (1994) The N‐terminal domain of a rab protein is involved in membrane–membrane recognition and/or fusion. EMBO Journal 13: 34–41.

Stoorvogel W, Geuze HJ, Griffith JM and Strous GJ (1988) The pathways of endocytosed transferrin and secretory protein are connected in the trans‐Golgi reticulum. Journal of Cell Biology 106: 1821–1829.

Tjelle TE, Brech A, Juvet LK, Griffiths G and Berg T (1996) Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation. Journal of Cell Science 109: 2905–2914.

Tulp A, Verwoerd D, Benham A and Neefjes J (1997) High‐resolution density gradient electrophoresis of proteins and subcellular organelles. Electrophoresis 18: 2509–2515.

Wei Ding LN, Zhang CY and Engelhardt JF (2006) rAAV2 traffics through both the late and recycling endosomes in a dose‐dependent fashion. Molecular Therapy 13: n.4.

Wilson RB and Murphy RF (1989) Flow cytometric analysis of endocytic compartments. Methods in Cell Biology 31: 293–317.

Wu CC, MacCoss MJ, Howell KE and Yates JR 3rd (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nature Biotechnology 21(5): 532–538.

Yates JR, Gilchrist A, Howell K and Bergeron J (2005) Proteomics of organelles and large cellular structures. Nature Reviews. Molecular Cell Biology 6: 702–714. 10.1038/nrm1711.

Zhang X, Fang A, Riley CP et al. (2010) Multi‐dimensional liquid chromatography in proteomics – a review. Analytica Chimica Acta 664(2): 101–113.

Further Reading

Rieder SE and Emr SD (2001) Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae. Current Protocols in Cell Biology, Chapter 3:Unit 3.8.

Weber PJ, Weber G and Eckerskorn C (2004) Isolation of organelles and prefractionation of protein extracts using free‐flow electrophoresis. Current Protocols in Protein Science, Chapter 22:Unit 22.5. DOI:10.1002/0471140864.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Guimaraes de Araujo, Mariana Eca, Fialka, Irene, and Huber, Lukas A(Dec 2010) Endocytic Organelles: Methods for Preparation and Analysis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002591.pub2]