Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

Abstract

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

“…The depletion of TPLATE was observed in parallel with a similar lack of enrichment of dynamin-related protein 1c (DRP1c), which functions outside the CCV (14). Critically, these finding have recently been confirmed by mass spectrometry of purified CCV samples in which none of the TPC members were found to be significantly enriched in CCVs (15). The absence of TPLATE enrichment in purified CCVs demonstrates that TPLATE is not incorporated within CCV structures as previously predicted, and when combined with the early departure of TPLATE from CCVs (Fig.…”

“…1 D and E). Notably, this result has been confirmed by mass spectrometry of purified CCVs, in which it was found that all members of the TCP failed to show any strong enrichment in CCVs (15). Thirdly, to increase our spatial resolution of live CME events, we used SIM.…”

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

“…By using the same sample preparation for biochemically isolated CCVs, we could directly compare them to specific CCV populations visualized inside cells. While we found that there were small differences in the sizes of endocytic and isolated CCVs, this is likely due to the fact that the isolated CCV preparations from whole cell lysates contain a mixture of CCVs derived from different origins -clathrin-mediated endocytosis and the early endosome/trans-Golgi network (22,27,28). We then validated our high throughput pseudo 3D analysis by applying it to metal replicas of unroofed cells carrying an inducible loss of endocytic membrane bending.…”

An Update on Coat Protein Complexes for Vesicle Formation in Plant Post-Golgi Trafficking

“…This raises out another question: how do AP1 and AP4 distinguish their specific cargoes on TGN/EE in two independent pathways or whether they function in parallel with overlapping cargos? In another finding using tandem mass spectrometry analysis, only AP1, AP2, and AP4 subunits were identified in enriched CCV proteome in Arabidopsis (Dahhan et al, 2021). It is possible that other accessory proteins might be involved to assemble as coatomer with similar function as the clathrin cage in AP3 vesicles.…”