The PA‐TM‐RING protein RING finger protein 13 is an endosomal integral membrane E3 ubiquitin ligase whose RING finger domain is released to the cytoplasm by proteolysis

Abstract

Proteins of the PA-TM-RING family have a proteaseassociated (PA) domain and a RING finger domain separated by a transmembrane (TM) domain. PA domains are 120-210 amino acid sequences located in the noncatalytic regions of diverse proteases [1,2] PA-TM-RING proteins have an N-terminal protease-associated domain, a structure found in numerous proteases and implicated in protein binding, and C-terminal RING finger and PEST domains. Homologous proteins include GRAIL (gene related to anergy in leukocytes), which controls T-cell anergy, and AtRMR1 (receptor homology region-transmembrane domain-RING-H2 motif protein), a plant protein storage vacuole sorting receptor. Another family member, chicken RING zinc finger (C-RZF), was identified as being upregulated in embryonic chicken brain cells grown in the presence of tenascin-C. Despite algorithm predictions that the cDNA encodes a signal peptide and transmembrane domain, the protein was found in the nucleus. We showed that RING finger protein 13 (RNF13), the murine homolog of C-RZF, is a type I integral membrane protein localized in the endosomal ⁄ lysosomal system. By quantitative real-time RT-PCR analysis, we demonstrated that expression of RNF13 is increased in adult relative to embryonic mouse tissues and is upregulated in B35 neuroblastoma cells stimulated to undergo neurite outgrowth. We found that RNF13 is very labile, being subject to extensive proteolysis that releases both the protein-associated domain and the RING domain from the membrane. By analyzing microsomes, we showed that the ectodomain is shed into the lumen of vesicles, whereas the C-terminal half, which possesses the RING finger, is released to the cytoplasm. This C-terminal fragment of RNF13 has the ability to mediate ubiquitination. Proteolytic release of RNF13 from a membrane anchor thus provides unique spatial and temporal regulation that has not been previously described for an endosomal E3 ubiquitin ligase.Abbreviations APP, Alzheimer's precursor protein; AtRMR1, Arabidopsis thaliana receptor homology region-transmembrane domain-RING-H2 motif protein; CHO, Chinese hamster ovary; C-RZF, chicken RING zinc finger; CTF, cytoplasmic C-terminal fragment; EEA1, early endosomal antigen 1; ER, endoplasmic reticulum; GRAIL, gene related to anergy in leukocytes; HA, hemagglutinin; HAF, hemagglutinin and 3· FLAG epitopes; HRP, horseradish peroxidase; ICD, intracellular domain; LAMP2, lysosomal-associated membrane protein 2; MPR, mannose 6-phosphate receptor; MVB, multivesicular body; NLS, nuclear localization signal; PA, protease-associated; PDI, protein disulfide isomerase; PNGase F, peptide: N-glycosidase F; RNF13, RING finger protein 13; TM, transmembrane.

The RING finger E3 ligase STRF1 is involved in membrane trafficking and modulates salt‐stress response in Arabidopsis thaliana

“…Indeed, other RING E3 ligases similar to STRF1 location have been identified in mammals. They include, among others, Tal1, which mediates Tsg101 ubiquitination and cargo sorting into vesicles (Amit et al, 2004) and RNF13, which localizes to the endosome membrane, and may take part in the ubiquitin-mediated endosome/lysosome recycling system (Bocock et al, 2009(Bocock et al, , 2010(Bocock et al, , 2011. Until now, only one E3 ligase from Arabidopsis, KEG (KEEP ON GOING), has been reported to localize to the TGN/EE, and to regulate endocytic trafficking and/or the formation of signaling complexes on TGN/EE vesicles during stress responses (Gu and Innes, 2011).…”

Mechanisms and Concepts Paving the Way towards a Complete Transport Cycle of Plant Vacuolar Sorting Receptors

“…This hypothesis has not been evaluated by direct experiments, but the RING domain in the C terminus of RMR is highly conserved among E3 ubiquitin protein ligases. A mammalian type I membrane spanning protein with a similar domain structure as plant RMRs (RNF13) has recently been shown to localize to endosomes where it is rapidly proteolysed to release the lumenal N-terminal domain as well as a cytoplasmic fragment that can mediate ubiquitination (Bocock et al, 2009). Lumenal release of the N-terminal domain may also happen for plant RMRs, as shown by an RFP-RMR fusion protein containing the TM domain and cytosolic tail of At-RMR1, which leads to accumulation of soluble RFP in the central vacuole lumen (Scabone et al, 2011).…”

The Role of the Transmembrane RING Finger Proteins in Cellular and Organelle Function

“…Thus, RNF13 may contribute to myogenesis as a negative regulator of cell proliferation. In addition, RNF13 expression is increased in differentiating rat B35 neuroblastoma cells, and overexpression of RNF13 promotes neurite extension in rat PC12 pheochromocytoma cells, suggesting an involvement of RNF13 in neuronal development [125]. High-level expression of RNF13 is also observed in pancreatic ductal adenocarcinoma as well as in precancerous pancreatic lesions, suggesting its involvement in cancer development [126].…”

“…RNF13 has been reported to be localized to various membrane structures, including the nucleus, ER, Golgi apparatus and endosomes. Recent studies have reported that RNF13 is present in the endosomal and lysosomal compartments, and the C-terminal region containing the RNF domain is released into the cytosol by proteolytic cleavage [125]. Activation of protein kinase C inhibits this cleavage and stabilizes the full-length RNF13 [127].…”