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.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
Jeffrey P
JP
Bocock
Stephanie
S
Carmicle
Egbert
E
Madamba
Ann H
AH
Erickson
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000201:enzymology*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D016253:Microscopy, Immunoelectron / Q000379:methods; D061988:Proteasome Inhibitors; D017434:Protein Structure, Tertiary; D011990:Receptors, Transferrin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D061988:Proteasome Inhibitors; D011990:Receptors, Transferrin; C540080:RNF13 protein, human; D044767:Ubiquitin-Protein Ligases; D013755:Tetradecanoylphorbol Acetate