Free‐Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes

Fuchs, Renate; Ellinger, Isabella

doi:10.1002/0471143030.cb0311s14

Cited by 2 publications

(1 citation statement)

References 21 publications

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“…Rab5 vesicles containing EYFP-Rab5a were prepared as described [30] . Briefly, HepG2 cells expressing pSin-EYFP-Rab5a were starved for 24 hours before being scraped and pelleted at 4°C.…”

Section: Methodsmentioning

confidence: 99%

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

Zhang

Gao

et al. 2011

PLoS ONE

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Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4) and Endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H+)-ATPases (V-ATPases) to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

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“…Rab5 vesicles containing EYFP-Rab5a were prepared as described [30] . Briefly, HepG2 cells expressing pSin-EYFP-Rab5a were starved for 24 hours before being scraped and pelleted at 4°C.…”

Section: Methodsmentioning

confidence: 99%

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

Zhang

Gao

et al. 2011

PLoS ONE

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Endocytic Organelles: Methods for Preparation and Analysis

Araújo

Fialka

Huber

2010

Encyclopedia of Life Sciences

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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.

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Free‐Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes

Cited by 2 publications

References 21 publications

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

Endocytic Organelles: Methods for Preparation and Analysis

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