Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver.
We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Finally we identified 21 CCV-associated proteins in liver not yet characterized in mammals. Our results further validate the peptide accounting approach, reveal new information on the properties of CCVs, and allow for the use of quantitative proteomics to compare abundant components of organelles under different experimental and pathological conditions.
Girard
Martine
M
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Québec H3A 2B4, Canada.
Allaire
Patrick D
PD
McPherson
Peter S
PS
Blondeau
Francois
F
D000818:Animals; D001921:Brain / Q000378:metabolism*; D033922:Clathrin Heavy Chains / Q000378:metabolism*; D033941:Clathrin Light Chains / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D013347:Subcellular Fractions; D013572:Synaptic Vesicles / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism
D033941:Clathrin Light Chains; D004268:DNA-Binding Proteins; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D033922:Clathrin Heavy Chains