Proximity labeling proteomics reveals critical regulators for inner nuclear membrane protein degradation in plants

Aobo Huang,Aobo Huang,Yu Tang,Xuetao Shi,Xuetao Shi,Xiaohan Yan,Xiaohan Yan,Min Jia,Huiqin Chen,Huiqin Chen,Jinheng Zhu,Yangnan Gu,Yangnan Gu

doi:10.1038/s41467-020-16744-1

Abstract

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.

Highlights

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; overaccumulation of INM proteins results in a range of rare genetic disorders
To identify candidates that are associated with SUN1 at the INM and exclude those that associate with both the INM and the outer nuclear membrane (ONM), we reanalyzed the SUN1 PLLFQMS data using the ONM-anchored protein WIT126 as a control (Fig. 1a)
We identified a total of four WIT1specific preys, including WIP1, WIP3, RanGAP1, and RanGAP2 (Fig. 1c and Supplementary Fig. 2b), supporting previous reports that WIT1 forms complexes with WIP proteins and anchors RanGAPs to the ONM26,29,30

Summary

Introduction

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; overaccumulation of INM proteins results in a range of rare genetic disorders. Via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We report a group of periphery NE proteins that are explicitly associated with the INM and involved in ubiquitinmediated proteolysis They include CDC48 proteins, its cofactors UFD1 and NPL4, and a specific subgroup of plant ubiquitin regulatory X (UBX) domain-containing proteins (PUXs). We showed that the CDC48 activity at the INM may be directly regulated by a specific subgroup of PUX proteins, including PUX3, PUX4, and PUX5 These PUX proteins have evolved a membrane preference for the INM through association with the nucleoskeleton and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants

Methods

Results

Conclusion