Masking of a Nuclear Signal Motif by Monoubiquitination Leads to Mislocalization and Degradation of the Regulatory Enzyme Cytidylyltransferase

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 (K-57) 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 K-57R 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.