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Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, rhomboid-like protein 4 (RHBDL4) is an endoplasmic reticulum (ER)-resident protease that forms a ternary complex with ubiquitinated substrates and p97/valosin-containing protein (VCP), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the preT-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein (APP) to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α (TGFα) in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. Protein tyrosine phosphatase non-receptor type 1 (PTP1B alias PTPN1) was also identified as potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys-63-linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

Original publication

DOI

10.1074/jbc.RA118.007074

Type

Journal article

Journal

J Biol Chem

Publication Date

07/06/2019

Keywords

BioID, K63-linked ubiquitin, PTP1B PTP1N, VCP p97, endoplasmic-reticulum-associated protein degradation (ERAD), intramembrane proteolysis, phosphoproteomics, polyubiquitin chain, protein-protein interaction, proteostasis, rhomboid protease, rhomboid-like protein 4 (RHBDL4), tyrosine-protein phosphatase (tyrosine phosphatase)