By Dana A. Dahhan and Sebastian Y. Bednarek, Dept. of Biochemistry, UW-Madison
Background: Trafficking of vacuolar and plasma membrane proteins and other macromolecules is essential for plant development, nutrient uptake, stress responses, and pathogen defense. At the trans-Golgi network/early endosomes and plasma membrane, the oligomerization of repeating clathrin protein units together with varying complements of additional accessory proteins generate a membrane coat that sequesters cargo such as membrane, proteins, and extracellular ligands into transport vesicles. The identities of these clathrin-associated proteins remain to be fully elucidated, but are of critical importance as they facilitate cargo transport in a directional manner, thereby modulating cargo abundance at subcellular sites and consequently, cargo protein function.
Question: What proteins are associated with clathrin-coated vesicles (CCVs) isolated from plant cells?
Findings: CCVs were enriched by differential centrifugation from suspension-cultured Arabidopsis cells and analyzed by unlabeled shotgun proteomics using two parallel methodologies to establish a core proteome of plant CCVs. In addition, a differential stable isotope labeling strategy combined with immunoblot analyses were used to quantitatively assess the enrichment or depletion of proteins with the purified CCVs. Manual annotation of proteins that were ≥ two-fold enriched identified conserved proteins involved in CCV-mediated trafficking between plants and other eukaryotes as well as key evolutionary divergences including the association of the adaptor, AP-4, with plant CCVs. The identities and metrics of proteins associated with plant CCVs provide a key reference for researchers studying eukaryotic membrane trafficking pathways mediated by the formation and transport of CCVs.
Next steps: Future research will investigate the involvement of CCV-associated proteins in endocytosis and post-Golgi trafficking and assess changes in the dynamics and regulation of clathrin-mediated endocytic and post-Golgi trafficking.
Dana A. Dahhan, Gregory D. Reynolds, Jessica J. Cárdenas, Dominique Eeckhout, Alexander Johnson, Klaas Yperman, Walter A. Kaufmann, Nou Vang, Xu Yan, Inhwan Hwang, Antje Heese, Geert De Jaeger, Jiri Friml, Daniel Van Damme, Jianwei Pan, Sebastian Y. Bednarek. (2022). Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components. https://doi.org/10.1093/plcell/koac071