Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development. KEY WORDS: Developmental signaling, Endocytic machinery, ESCRT-0, Hrs, Ubpy, Drosophila INTRODUCTIONMany developmental signaling events are initiated through ligand binding to respective receptors at the plasma membrane. Following uptake into endocytic vesicles referred to as early or sorting endosomes, membrane receptors may recycle back to cell surface via tubular recycling endosomes. However, when directed by a ubiquitin (Ub) signal, receptor complexes are captured and sorted towards lysosomal degradation (Henne et al., 2011). The Ub moieties of protein cargos are recognized by ESCRT complexes, and are sorted into invaginating multivesicular bodies (MVBs). Mature MVBs fuse with lysosomes to deliver protein cargos for degradation (Raiborg and Stenmark, 2009). ESCRT-0 is composed of heterodimers of two subunits: Hrs and Stam. Both subunits bind Ub and clathrin, and Hrs additionally has an FYVE zinc-finger domain that binds phosphatidylinositol 3-phosphate. The ability to bind both lipid and Ub allows ESCRT-0 to initiate endosomal sorting (Clague et al., 2012).Ubpy (or USP8), a USP family deubiquitinase (DUB), participates in sorting of ubiquitylated receptors through its interaction with ESCRT-0 (Mizuno et al., 2005;Row et al., 2006). Most studies of Ubpy focus on endosomal trafficking of growth factor receptor tyrosine kinases (RTKs) in cultured vertebrate cells. However, conflicting data have been reported. In some cases, reduced Ubpy activity results in accumulation of ubiquitylated cargos (Bowers et al., 2006;Mizuno et al., 2006;Row et al., 2006; Alwan and van Leeuwen, 2007). Other studies suggest that Ubpy promotes RTK stability (Mizuno et al., 2005;Niendorf et al., 2007; Berlin et al., 2010). The role of Ubpy in Drosophila development is equally controversial. It was ...
Protein O-GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O-GlcNAcylated proteins have been identified in animals, the plant O-GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O-GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both Nazidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz) can metabolically label O-GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP-galactose 4epimerases were found to interconvert UDP-GalNAz and UDP-GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide-incorporated O-GlcNAc with alkyne-biotin via click chemistry, the O-GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O-GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O-GlcNAcylated proteins were enriched in various plant-specific processes such as hormone responses. By coexpression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O-GlcNAc transferase, we validated that the MGL-identified proteins were O-GlcNAcmodified. Our work establishes a powerful tool for profiling plant O-GlcNAylation and provides an invaluable resource for investigating the functional role of O-GlcNAc in Arabidopsis.
Summary O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) is a ubiquitous post‐translation modification occurring in both animals and plants. Thousands of proteins along with their O‐GlcNAcylation sites have been identified in various animal systems, yet the O‐GlcNAcylated proteomes in plants remain poorly understood. Here, we report a large‐scale profiling of protein O‐GlcNAcylation in a site‐specific manner in rice. We first established the metabolic glycan labelling (MGL) strategy with N‐azidoacetylgalactosamine (GalNAz) in rice seedlings, which enabled incorporation of azides as a bioorthogonal handle into O‐GlcNAc. By conjugation of the azide‐incorporated O‐GlcNAc with alkyne‐biotin containing a cleavable linker via click chemistry, O‐GlcNAcylated proteins were selectively enriched for mass spectrometry (MS) analysis. A total of 1591 unambiguous O‐GlcNAcylation sites distributed on 709 O‐GlcNAcylated proteins were identified. Additionally, 102 O‐GlcNAcylated proteins were identified with their O‐GlcNAcylation sites located within serine/threonine‐enriched peptides, causing ambiguous site assignment. The identified O‐GlcNAcylated proteins are involved in multiple biological processes, such as transcription, translation and plant hormone signalling. Furthermore, we discovered two O‐GlcNAc transferases (OsOGTs) in rice. By expressing OsOGTs in Escherichia coli and Nicotiana benthamiana leaves, we confirmed their OGT enzymatic activities and used them to validate the identified rice O‐GlcNAcylated proteins. Our dataset provides a valuable resource for studying O‐GlcNAc biology in rice, and the MGL method should facilitate the identification of O‐GlcNAcylated proteins in various plants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.