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Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
Published on: October 20, 2014
Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics
B Ritter1, F Blondeau, A Yu Denisov
1Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 University St., Montreal, QC, Canada.
Biochemical Society Transactions
|October 21, 2004
Summary
Subcellular proteomics identified novel proteins in brain clathrin-coated vesicles (CCVs), revealing their role in synaptic vesicle recycling and CCV formation. This approach enhances understanding of cellular compartment functions.
Area of Science:
- Cellular Biology
- Proteomics
- Neuroscience
Background:
- Subcellular proteomics combines fractionation and mass spectrometry to identify proteins within cellular compartments.
- Previous studies have identified known and novel proteins in various organelles.
- Clathrin-coated vesicles (CCVs) are crucial for intracellular trafficking.
Purpose of the Study:
- To analyze the protein components of CCVs isolated from the adult brain using subcellular proteomics.
- To quantitatively assess protein complexes associated with CCVs.
- To identify novel proteins involved in CCV formation and function.
Main Methods:
- Subcellular fractionation of adult brain tissue.
- Mass spectrometry (MS) based proteomics to identify and quantify proteins.
- Analysis of fragmented peptides for quantitative assessment of protein complexes.
- Characterization of novel identified proteins (NECAP1 and NECAP2).
Main Results:
- Identified known components of post-fusion synaptic vesicles, indicating CCVs recycle synaptic vesicles in the brain.
- Discovered novel proteins participating in CCV formation at the trans-Golgi network and plasma membrane.
- Characterized NECAP1 and NECAP2, identifying a new motif for interaction with the clathrin adaptor protein 2.
Conclusions:
- Brain CCVs play a significant role in synaptic vesicle recycling.
- Subcellular proteomics is a powerful tool for discovering novel proteins and functions of cellular compartments.
- The identified proteins and motifs provide new insights into CCV biogenesis and trafficking mechanisms.
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