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 (CCTalpha) is a proteolytically sensitive lipogenic enzyme containing an NH(2)-terminal nuclear localization signal (NLS). We show here that CCTalpha 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 CCTalpha-ubiquitin fusion protein that mimics the monoubiquitinated enzyme resulted in cytoplasmic retention. A CCTalpha K(57R) mutant exhibited an extended half-life, was retained in the nucleus, and displayed proteolytic resistance. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha 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.