Trafficking and proteolytic processing of RNF13, a model PA-TM-RING family endosomal membrane ubiquitin ligase.

RING finger protein 13 (RNF13) is a ubiquitously expressed, highly regulated ubiquitin ligase anchored in endosome membranes. A RING domain located in the cytoplasmic half of this type 1 membrane protein mediates ubiquitination in vitro but physiological substrates have not yet been identified. The protein localized in endosomal membranes undergoes extensive proteolysis in a proteasome-dependent manner, but the mRNA level can be increased and the encoded protein stabilized under specific physiological conditions. The cytoplasmic half of RNF13 is released from the membrane by regulatory proteases and therefore has the potential to mediate ubiquitination at distant sites independent of the full-length protein. In response to protein kinase C activation, the full-length protein is stabilized and moves to recycling endosomes and to the inner nuclear membrane, which exposes the RING domain to the nucleoplasm. Thus RNF13 is a ubiquitin ligase that can potentially mediate ubiquitination in endosomes, on the plasma membrane, in the cytoplasm, in the nucleoplasm or on the inner nuclear membrane, with the site(s) regulated by signaling events that modulate protein targeting and proteolysis.

Nuclear targeting of an endosomal E3 ubiquitin ligase.

Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

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.

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.

Targeting to lysosomes in mammalian cells: the biosynthetic and endocytic pathways.

Endogenous or ectopically expressed lysosomal proteins can be detected in their biosynthetic or endocytic pathways by Western blotting of biosynthetic forms in cells, cell fractions, or their culture medium, by pulse-chase radiolabeling accompanied by immunoprecipitation, or by electron or immunofluorescence microscopy. Western blotting and microscopy reveal the steady-state distribution of a protein, whereas pulse-chase studies are required both to identify transient forms and to define the relationship of the biosynthetic forms detected. Targeting to lysosomes can be dramatically affected by synthesis levels and carbohydrate modification, whether the synthesis is upregulated naturally, for example, by cell transformation, or whether it results from ectopic expression. This occurs because a lysosomal protein, unlike a protein expressed in the cytoplasm, must interact with receptors and be packaged into vesicles that mediate its transport though the secretory pathway. Use of microscopy to establish localization is, therefore, a key aspect of characterization of the cellular pathways utilized by lysosomal proteins.

Biosynthesis and alternate targeting of the lysosomal cysteine protease cathepsin L.

Upregulation of cathepsin L expression, whether during development or cell transformation, or mediated by ectopic expression from a plasmid, alters the targeting of the protease and thus its physiological function. Upregulated procathepsin L is targeted to small dense core vesicles and to the dense cores of multivesicular bodies, as well as to lysosomes and to the plasma membrane for selective secretion. The multivesicular vesicles resemble secretory lysosomes characterized in specialized cell types in that they are endosomes that stably store an upregulated protein and they possess the tetraspanin CD63. Morphologically the multivesicular endosomes also resemble late endosomes, but they store procathepsin L, not the active protease, and they are not the major site for LAMP-1 accumulation. Distinction between the lysosomal proenzyme and active protease thus identifies two populations of multivesicular endosomes in fibroblasts, one a storage compartment and one an enzymatically active compartment. A distinctive targeting pathway using aggregation is utilized to enrich the storage endosomes with a particular lysosomal protease that can potentially activate and be secreted.

Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L.

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nM, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu.