Non-stoichiometric Relationship between Clathrin Heavy and Light Chains Revealed by Quantitative Comparative Proteomics of Clathrin-coated Vesicles from Brain and Liver

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. Vesicle budding and trafficking via clathrin-coated pits (CCPs) 1 and vesicles (CCVs) provides a major route by which proteins are transported out of the trans-Golgi network (TGN) and by which receptors, transporters, and nutrients are endocytosed at the plasma membrane (1-3). Many clathrin-dependent trafficking events mediate cargo transport that is needed in all cell types. These "housekeeping" forms of clathrin trafficking include the turnover of plasma membrane proteins and lipids, endocytic uptake of nutrients such as ironsaturated transferrin and low density lipoproteins, and endocytosis of a diverse range of activated growth factor receptors (1-3). Moreover all cells have housekeeping trafficking at the TGN. An important example is the delivery of mannose 6-phosphate-tagged lysosomal hydrolases from the TGN to endosomes/lysosomes via the mannose 6-phosphate receptor (MPR) (4).In addition to these housekeeping activities of CCVs, some tissues have specialized trafficking needs. For example, in secretory cells, clathrin coats are involved in the formation of secretory granules at the TGN (5), and polarized cells utilize CCVs for the trafficking of certain receptors from the TGN to the basolateral membrane necessary for the maintenance of polarity (2). At the plasma membrane, intestinal epithelial cells in rat or placental cells in humans use CCVs for the uptake of maternal immunoglobulins, a necessary aspect of maternal derived immunity (6). A striking example of specialized CCV function is seen in neurons, which communicate by releasing neurotransmitters through fusion of synaptic vesicles with the plasma membrane following transient increases in Ca 2ϩ concentration (7). These vesicles are then retrieved through CCVs (8 -10). Thus, neurons need CCVs not only for housekeeping forms of clathrin-mediated endocytosis but also to retrieve synaptic vesicle membranes. It has been unclear whether or not the mechanisms mediating these two related but distinct events taking place at the plasma membrane could be distinguished. Moreover the relative amount of brain CCVs specialized for synaptic function has never been assessed.The presence of clathr...

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

“…Since several hundreds of proteins can be identified for each sample, large numbers of peptide spectrum matches (often several hundred) are collected for each individual protein, which allows for accurate relative quantitation of distinct HCPs across samples via spectral counting, a simple and accurate label-free quantitation method. [23][24][25][26][27][28][29][30][31] The identification of large numbers of proteins per sample also enabled multivariate statistical analysis, which like spectral counting, relies on large numbers of data points to yield reliable results. Since many samples, generated from a variety of different cell lines and processes, were analyzed in this study, a streamlined nomenclature was adopted for sample differentiation.…”

The DnaJ-domain Protein RME-8 Functions in Endosomal Trafficking

“…Identification of Mammalian RME-8-A proteomic analysis of CCVs isolated from rat liver identified proteins that are predicted products of cDNAs and genomic sequence (24). One such protein, identified in the annotated rat genome under gi27721389, is the rat homolog of RME-8.…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…The principal components of coated vesicles isolated from various sources are the heavy and light chains of clathrin and the four subunits of the heterotetrameric "adaptor complexes" (Kirchhausen 2000;Blondeau et al 2004;Girard et al 2005;Borner et al 2006Borner et al , 2012. The heterotetrameric adaptors link the clathrin coat and the membrane bilayer, and they are the principal cargo-recognition molecules.…”