Proteome of clathrin-coated vesicles purified from A. thaliana suspension-cultured cells

ABSTRACT: In Eukaryotes, clathrin coated vesicles (CCVs) facilitate the internalization of material from the cell surface via clathrin-mediated endocytosis (CME), as well as the movement of cargo in post-Golgi trafficking pathways. Key to our 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 clathrin coated vesicles. CCVs were purified from undifferentiated Arabidopsis suspension cultured cells through a differential centrifugation scheme (Reynolds, et al. Methods Mol Biol. 2014). We undertook three proteomic analyses of purified CCVs. In the first (Dataset A), four biological replicates of purified CCVs were separated by 1D SDS-PAGE before in-gel digestion with trypsin and shotgun proteomic identification of the CCV associated proteins. In the second (Dataset B), heavy and light formaldehyde labels were applied to the deuterium/Ficoll gradient load (DFGL) and final CCV fractions in a reciprocal manner across two independent biological replicates. Fractions were separated by 1D SDS-PAGE prior to gel sectioning and in-gel digestion with trypsin before reaction of primary amines with heavy and light labels. Previous work has demonstrated that the enrichment of CCV-associated proteins in the differential centrifugation scheme used to purify CCVs is greatest in the penultimate step, a deuterium/Ficoll gradient, so the use of heavy and light formaldehyde enabled the determination of enriched and depleted proteins as CCVs were purified. In the third (Dataset C) experiment, three biological replicates were not separated by SDS-PAGE and were digested in solution with trypsin before shotgun proteomic identification of CCV associated proteins. 3,548 proteins were identified in the first proteomic analysis in two or more replicates. 1,109 proteins were identified between both biological replicates in the dimethyl labeling experiment. 1,981 protein groups were identified in at least two of three biological replicates from the proteomics methodology lacking separation of CCVs by SDS-PAGE. 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.

INSTRUMENT(S): ThermoFisher LTQ Linear Ion Trap, Q Exactive HF, ThermoFisher LTQ FT-ICR Ultra, Q Exactive

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Sebastian Y. Bednarek  

PROVIDER: MSV000087472 | MassIVE | Thu May 20 21:22:00 BST 2021

SECONDARY ACCESSION(S): PXD026180

REPOSITORIES: MassIVE