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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 assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex 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 TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) 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.
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CALM supports clathrin-coated vesicle completion upon membrane tension increase
Significance Clathrin-coated vesicles (CCVs) are endocytic carriers responsible for the internalization of receptor-bound ligands and extracellular fluids. CCV assembly occurs by the sequential recruitment of clathrin, adaptors, and other accessory molecules that promote curvature formation. To form, CCVs need to overcome the local plasma membrane tension that varies during cell cycle, development, and cell polarization. Using quantitative fluorescence microscopy, we demonstrate that the adaptor CALM is a major determinant of CCV formation upon membrane tension increase. Since CALM is differentially expressed, our results demonstrate that competence in clathrin-mediated endocytosis is tissue specific, providing mechanistic explanation why CALM depletion strongly affects embryo development and red blood cell differentiation with minor effects in other systems.
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- Award ID(s):
- 1751113
- PAR ID:
- 10251508
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 25
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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