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Vol. 19, Issue 11, 4588-4601, November 2008

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Lysosomal Localization of Ubiquitinated Jun Requires Multiple Determinants in a Lysine-27–Linked Polyubiquitin Conjugate

Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0650

Submitted May 19, 2008; Revised July 14, 2008; Accepted August 12, 2008
Monitoring Editor: William P. Tansey

Ubiquitination regulates many cellular functions, including protein localization and degradation. Each function is specified by unique determinants in the conjugate. Ubiquitinated Jun is localized to lysosomes for degradation. Here, we characterized determinants of Jun ubiquitination and lysosomal localization by using ubiquitin-mediated fluorescence complementation (UbFC) in living cells and analysis of the stoichiometry of ubiquitin linked to Jun extracted from cells. The region of Jun and isoleucine-44 in ubiquitin were required for lysosomal localization of the conjugate. Ubiquitin containing only lysine-27, but no other single-lysine ubiquitin, mediated Jun ubiquitination, albeit at lower stoichiometry than wild-type ubiquitin. These conjugates were predominantly nuclear, but coexpression of lysine-27 and lysine-less ubiquitins enhanced the mean stoichiometry of Jun ubiquitination and lysosomal localization of the conjugate. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and tumor susceptibility gene 101 (TSG101) colocalized with ubiquitinated Jun. Knockdown of HRS or TSG101 inhibited lysosomal localization of ubiquitinated Jun and reduced Jun turnover. Ubiquitination of other Fos and Jun family proteins had distinct effects on their localization. Our results indicate that Jun is polyubiquitinated by E3 ligases that produce lysine-27–linked chains. Lysosomal localization of the conjugate requires determinants in Jun and in ubiquitin that are recognized in part by TSG101 and HRS, facilitating selective translocation and degradation of ubiquitinated Jun.

* Present address: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD 20892-2785.

Address correspondence to: Tom K. Kerppola (kerppola{at}umich.edu)

Abbreviations used: UbFC, ubiquitin-mediated fluorescence complementation; YFP, yellow fluorescent protein; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; TSG101, tumor susceptibility gene 101; Ub_n, ubiquitin chain composed of n protomers; VN, residues 1-155 of Venus fluorescent protein; CC, residues 156-238 of cyan fluorescent protein.