Unraveling Plant Nuclear Envelope Composition Using Proximity Labeling Proteomics

Abstract

The nuclear envelope (NE) defines the eukaryotic cell and functions in a myriad of fundamental cellular processes including but not limited to signal transduction, lipid metabolism, chromatin organization, and nucleocytoplasmic transportation. Although the general structure of the NE is well-conserved across eukaryotic kingdoms, its composition and functions vary substantially between species and remain largely unknown in plants. In this chapter, we describe a proximity-labeling-based proteomic approach to profile novel NE components in the model organism Arabidopsis. This method is generally suitable for the identification of protein components in subcellular compartments or protein complexes that are poorly accessible to traditional mass spectrometry approaches and can be easily applied to other plant species. In addition to giving a step-by-step detailed description of the proximity labeling proteomics procedure in plant samples, we also provide guidelines on the appropriate use of controls and statistical analysis to achieve a highly specific selection of probed candidates.

Keywords: Affinity purification; Biotinylation; Label-free quantification; Mass spectrometry; Nuclear envelope; Proximity labeling proteomics; TurboID.