PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

Abstract

The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

New Insights Into the Role of Seed Oil Body Proteins in Metabolism and Plant Development

“…These distinct motifs designate oleosins toward different degradation pathways according to the ubiquitination type. Deruyffelaere et al further confirmed that PUX10 localized to OBs interacts directly with ubiquitinated oleosins and mediates dislocation of oleosins by the AAA ATPase CDC48A (Deruyffelaere et al, 2018;Kretzschmar et al, 2018).…”

“…PUX10 localizes to OBs and binds to the ubiquitinated oleosins via its hydrophobic domain and interacts with ubiquitin and CDC48A via its UBA and UBX domains, respectively. As an adaptor, PUX10 recruits CDC48A to ubiquitinated oleosins, leading to dislocation of oleosins from OBs via the segregase activity of CDC48A (Deruyffelaere et al, 2018;Kretzschmar et al, 2018).…”

“…But so far, little information concerning the identity of the endogenous protease has been revealed. In addition to oleosin, caleosin, and steroleosin, several other OB-associated proteins (such as OBAP1 identified in maize scutellum) have been detected recently (López-Ribera et al, 2014;Müller et al, 2017;Kretzschmar et al, 2018;De Chirico et al, 2018). Additionally, distinct populations of OBs have been found to exist in plants, each possessing different proteins, suggesting functional differentiation of OBs in plant seedlings.…”

The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

“…and K63-linked diubiquitin K63Ub2) allowing addressing this protein to the 26S proteasome in the cytosol for its degradation [127]. It has been recently shown, by two independent teams, in the pollen tube of Nicotiana tabacum and in germinating seedlings of Arabidopsis thaliana, that the protein PUX10 (Plant UBX Domain-containing Protein 10), located on the surface of LDs, participated in the recognition of polyubiquitinated K48 motifs with its UBA domain [127,128]. PUX10, via its UBX domain, recruits the AAA-type ATPase Cell Division Cycle 48 protein (CDC48), a protein involved in the ERAD (Endoplasmic-reticulum-associated protein degradation) pathway.…”

Large-scale identification of ubiquitination sites on membrane-associated proteins inArabidopsis thalianaseedlings

“…Oleosin family protein (Deruyffelaere et al, 2015;Kretzschmar et al, 2018) AT3G01670 Unknown protein AT3G02130 RPK2/TOAD2/CLI1 AT3G03050 CSLD3/KJK/ATCSLD3 (Gu et al, 2016) AT3G03310 ATLCAT3 AT3G04120 GAPC/GAPC-1 (Peralta et al, 2016) AT3G04840 Ribosomal protein S3Ae (Kim et al, 2013;Svozil et al, 2014;Walton et al, 2016) Heat shock protein 70 (Hsp 70) family protein (Walton et al, 2016;Romero-Barrios et al, 2020) AT3G09740 SYP71 (Walton et al, 2016;Romero-Barrios et al, 2020) AT3G09840 CDC48,/CDC48A (Kim et al, 2013;Walton et al, 2016…”