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

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.

Recent advances in understanding the biological roles of the plant nuclear envelope

“…PUX3-5 were additionally shown to interact with CRWN4 and KAKU4. Treatment with a proteasome inhibitor, MG132, resulted in an increase in SUN1 accumulation, confirming SUN1 is a target of proteasome degradation98 . Loss-of-function mutants for PUX3-5 resulted in an increase in SUN1protein, suggesting PUX3-5 negatively regulate SUN1 degradation98 .…”

“…Treatment with a proteasome inhibitor, MG132, resulted in an increase in SUN1 accumulation, confirming SUN1 is a target of proteasome degradation98 . Loss-of-function mutants for PUX3-5 resulted in an increase in SUN1protein, suggesting PUX3-5 negatively regulate SUN1 degradation98 . It remains to be determined if PUX3-5 interface with and regulate INM proteins generally, or SUN proteins specifically and what role the interaction with CRWN4 and KAKU4 plays in this process.…”

“…A partial rescue of the opnr mutant results in enlarged nuclei in root cells, indicating a potential role for OPENER in nucleolar and nuclear size control97 .The BioID2 approach described above is also shedding first light on INM-specific protein degradation in plants. Among the interactors of SUN1, Huang et al identified CDC48, a chaperone involved in extracting membrane proteins for hydrolysis by the 26S proteasome, its cofactors UFD1 and NPL4, as well as a group of plant ubiquitin regulatory X domain-containing proteins (PUXs)98 . Five members of the CDC48 complex were identified as interactors of the INM proteins SUN1 and NEAP1: PUX3, PUX4, PUX5, UFD1B, and UFD1C.…”

Proximity labeling: an emerging tool for probing in planta molecular interactions

“…In 2019, Wong's group used BioID-based PL to identify the interaction partners of autophagy-related protein 8 (ATG8) and the 126 kDa replicase of tobacco mosaic virus (TMV) ( Das et al., 2019 ; Macharia et al., 2019 ). Very recently, Yangnan Gu's group used BioID2-based PL to profile the plant nuclear envelope and inner nuclear membrane proteomes in Arabidopsis ( Huang et al., 2020 ). They identified a novel component of the nuclear pore complex and unraveled critical regulators of inner nuclear membrane protein degradation, all of which enhance our understanding of the composition of subnuclear structures and the underlying functions of nuclear membrane-associated proteins ( Huang et al., 2020 ; Tang et al., 2020 ).…”

“…Very recently, Yangnan Gu's group used BioID2-based PL to profile the plant nuclear envelope and inner nuclear membrane proteomes in Arabidopsis ( Huang et al., 2020 ). They identified a novel component of the nuclear pore complex and unraveled critical regulators of inner nuclear membrane protein degradation, all of which enhance our understanding of the composition of subnuclear structures and the underlying functions of nuclear membrane-associated proteins ( Huang et al., 2020 ; Tang et al., 2020 ). These studies reinforced the robustness of PL methods for identifying membrane-associated interaction partners, which are inaccessible by traditional approaches, and indicated that PL methods could also be used to investigate global sub-organelle proteomes in plant systems as in mammalian cells ( Kim et al., 2016 ; Cho et al., 2020 ).…”