Abstract
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.
Highlights
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
Enrichment for protein bands corresponding to the molecular masses of clathrin heavy chain (CHC), clathrin light chains (CLCs), and the ␣, , ␥- and -adaptin subunits of the adaptor protein (AP)-1 and AP-2 complexes was observed in the consecutive fractions of the CCV preparation (Fig. 1A)
Numerous organelles and suborganellar compartments have been analyzed by subcellular proteomics, and in almost all cases, novel proteins have been identified, and the global analysis of the organelle has provided insights into organelle function that may not have been possible from the analysis of a smaller subset of the proteins (34 –36)
Summary
Antibodies—Monoclonal antibodies for CHC, AP-1 (␥-adaptin), and AP-2 (␣-adaptin) were from BD Biosciences. Preparation and Analysis of Liver CCVs—Liver CCVs were isolated using procedures described previously [21, 22] from adult rats that had been starved overnight. Peptides from the entire lane were grouped based on their GI number and defined as specific peptides for their cognate protein To add another level of confidence, only proteins found in two of three preparations and with five or more peptides were retained [26]. CCVs were isolated from the livers of starved adult rats using the same protocol. Variable amounts of protein from the resulting microsomal (P2) fractions from each tissue were analyzed by SDSPAGE and Western blot to generate an equivalent signal for the CHC. Gels were loaded with 100 g of microsomes prepared from a single tissue and 200 g of mixed tissues to account for the dilution of the tissues when mixed together
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
