Tài liệu Báo cáo khoa học: The PA-TM-RING protein RING finger protein 13 is an endosomal integral

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

Jeffrey P. Bocock1

, Stephanie Carmicle1

, Saba Chhotani1

, Michael R. Ruffolo1

, Haitao Chu2 and

Ann H. Erickson1

1 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA

2 Department of Biostatistics, University of North Carolina, Chapel Hill, NC, USA

Proteins of the PA-TM-RING family have a protease￾associated (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]. They

are found in multiple members of MEROPS peptidase

Keywords

E3 ubiquitin ligase; neurite outgrowth;

protease-associated domain; proteolysis;

RNF13

Correspondence

A. Erickson, Department of Biochemistry

and Biophysics, CB 7260 GM, University of

North Carolina, Chapel Hill, NC 27599, USA

Fax: +1 929 966 2852

Tel: +1 919 966 4694

E-mail: [email protected]

(Received 1 November 2008, revised 23

December 2008, accepted 20 January 2009)

doi:10.1111/j.1742-4658.2009.06913.x

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 neu￾roblastoma 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 mem￾brane. 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 tempo￾ral 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.

1860 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS