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Molecular and Cellular Biology, June 2009, p. 3062-3075, Vol. 29, No. 11
0270-7306/09/$08.00+0     doi:10.1128/MCB.01824-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Masking of a Nuclear Signal Motif by Monoubiquitination Leads to Mislocalization and Degradation of the Regulatory Enzyme Cytidylyltransferase{triangledown} ,{dagger}

Bill B. Chen2 and Rama K. Mallampalli1,2,3*

Departments of Internal Medicine,1 Biochemistry,2 Department of Veterans Affairs Medical Center, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 522423

Received 1 December 2008/ Returned for modification 9 January 2009/ Accepted 16 March 2009

Monoubiquitination aids in the nuclear export and entrance of proteins into the lysosomal degradative pathway, although the mechanisms are unknown. Cytidylyltransferase (CCT{alpha}) is a proteolytically sensitive lipogenic enzyme containing an NH2-terminal nuclear localization signal (NLS). We show here that CCT{alpha} is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS, resulting in disruption of its interaction with importin-{alpha}, nuclear exclusion, and subsequent degradation within the lysosome. Cellular expression of a CCT{alpha}-ubiquitin fusion protein that mimics the monoubiquitinated enzyme resulted in cytoplasmic retention. A CCT{alpha} K57R mutant exhibited an extended half-life, was retained in the nucleus, and displayed proteolytic resistance. Importantly, by using CCT{alpha}-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCT{alpha} monoubiquitination masks its NLS, resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking and life span of a critical regulatory enzyme in vivo.

* Corresponding author. Mailing address: The University of Iowa, Pulmonary and Critical Care Division, C-33K, GH, Department of Internal Medicine, Iowa City, IA 52242. Phone: (319) 356-1265. Fax: (319) 335-6506. E-mail: rama-mallampalli{at}uiowa.edu

{triangledown} Published ahead of print on 30 March 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, June 2009, p. 3062-3075, Vol. 29, No. 11
0270-7306/09/$08.00+0     doi:10.1128/MCB.01824-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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