Endocytic Organelles: Methods for Preparation and Analysis

Abstract

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.

Studying the regulation of endosomal cAMP production in GPCR signaling

“…Endosomes are isolated by density gradient centrifugation (Guimaraes de Araujo, Fialka, & Huber, 2010; Stasyk et al, 2007, 2010). …”