Monoubiquitination has emerged as a means to target resident nuclear proteins for export into the cytoplasm (5
); however, the mechanisms have not been elucidated. In the present study, we provide the first evidence that protein ubiquitination masks the NLS of a key regulatory enzyme, CCTα, thereby leading to abrogation of its binding to importin-α, its cytosolic retention, and lysosomal degradation. This was demonstrated using positional hybrid mutant constructs of CCTα that varied in intermolecular spacing between the NLS and ubiquitin. We further demonstrate that CCTα is not polyubiquitinated but undergoes site-specific monoubiquitination as a means to regulate enzyme half-life in cells. Evidence in support of CCTα monoubiquitination includes the observations that (i) CCTα was effectively conjugated with single ubiquitin in vitro; (ii) the enzyme was detected in cells in a monoubiquitin-CCTα complex; and (iii) cellular levels accumulated after treatment with NH4
Cl, which impairs the clearance of monoubiquitinated substrates. The data also suggest that juxtapositioning of a ubiquitin acceptor site (K57
) near an exposed nuclear targeting sequence is efficiently exploited by cells to mislocalize an enzyme that is normally targeted for nuclear membrane activation. Thus, monoubiquitination may be an important homeostatic signal by which cells brand CCTα for degradation to maintain an optimal balance of membrane phospholipid.
The present study differs from our previous work, where we showed that TNF-α-induced CCTα degradation is attenuated with lactacystin, suggesting that the enzyme is also polyubiquitinated and processed within the proteasome (21
). However, that earlier study differs significantly from the present study with regard to experimental design, the concentrations and kinetics of several reagents used, and the culture conditions. Despite using clasto
-lactacystin in the present study, an active metabolite of lactacystin that is severalfold more active than the lactacystin used previously, we were not able to abrogate TNF-α-induced CCTα degradation. Our results do not exclude the possibility that CCTα is monoubiquitinated, multiubiquitinated, or even polyubiquitinated depending on experimental conditions as observed for other regulatory proteins (e.g., p53) or hormone receptors that are channeled via different degradative pathways (15
). TNF-α causes both polyubiquitination and monoubiquitination of IκB kinase proteins (4
), and the cytokine's ability to differentially regulate CCTα ubiquitination would not be surprising given its pleiotropic effects. Although we identified a physiologically relevant ubiquitin acceptor site (K57
) within CCTα, the CCTK57R
mutant significantly, but not completely, displayed reduced ability to be monoubiquitinated (Fig. ). Thus, there may be a second ubiquitination acceptor site within the enzyme. Preliminary mass spectrometry data showing that CCTK341
may be ubiquitinated is consistent with the detection of two immunoreactive CCTα bands at 50 and 58 kDa at times in cell lysates (data not shown). Thus, CCTα might be multiubiquitinated in vivo.
The CCTα NLS harbors a stretch of basic residues typical of nuclear signal motifs that might interact with the karyopherin, importin-α, that after forming a complex with CCT translocates through the nuclear pore complex; once within the nucleus, the karyopherin-cargo complex dissociates by the action of RanGTPase (26
). CCTα cytoplasmic-nuclear shuttling also appears to utilize this pathway since importin-α was bound to unmodified enzyme in vitro and in vivo, but less so with monoubiquitinated enzyme (Fig. ). Preliminary fluorescence recovery after photobleaching studies show that CFP-CCTα nuclear import is accelerated after cotransfection of cells with importin-α (see Movies S1 and S2 in the supplemental material). Moreover, posttranslational modifications, masking of cargo nuclear transport signals by conformational changes, and protein-protein interaction can prevent karyopherin-cargo binding and disrupt the trafficking of NLS-containing proteins (1
). The masking of nuclear transport signals by monoubiquitination, however, represents a previously unrecognized mechanism whereby proteins are retained within the cytoplasm.
Thus far, the most well-understood monoubiquitinated protein is p53, a protein ubiquitinated by the RING domain E3 ligase, MDM2; this monoubiquitination event causes p53 nuclear export (20
). The detailed mechanism of p53 nuclear export, however, remains largely unknown. A recent study hypothesized, but did not test, the possibility that p53 monoubiquitination within its carboxyl terminus alters its three-dimensional structure, unmasking a hidden carboxyl-terminal NES (5
). Unlike p53, CCTα does not harbor a canonical leucine-rich NES, nor does leptomycin, an inhibitor of exportin-α, lead to nuclear enzyme accumulation (24
). Thus, it would appear that the molecular model for CCTα trafficking by monoubiquitination would be NES independent and might involve factors that lead to its nuclear exclusion by functional disruption of its NLS. To address this, a ubiquitin-CCTα fusion protein (CFP-CCTK57
-Ub) was constructed. When a single ubiquitin copy is linked to Lys57
, the carboxyl-terminal ubiquitin Gly will form an isopeptide bond with the Lys
-amino group of CCTα. Because of the flexibility of the Gly residue, the CCTα-ubiquitin fusion protein will mimic monoubiquitinated CCTα. However, there are two caveats using this approach. First, ubiquitin fusions can themselves serve as ubiquitins and conjugate to other molecules (5
). Second, spacing between Ser56
within the CCTα amino terminus would normally be in the order of only 1 to 2 Å but, based on the crystal structure of ubiquitin, its insertion between these residues is predicted to increase this distance to 20 to 30 Å, thus potentially altering CCTα conformation. Nevertheless, since residues 8 to 28 representing the CCTα NLS would be in close proximity to the ubiquitinated moiety, we hypothesized that in the CFP-CCTK57
-Ub fusion protein the NLS is masked. To verify this, we shifted the NH2
-terminal segment (aa 1 to 40) of CCTα to the carboxyl-terminal end to increase the intermolecular distance between the NLS and ubiquitin (Fig. ). Indeed, expression of this ubiquitin fusion construct (CFP-CCTΔN40
) was sufficient to trigger nuclear import of CCTα by exposure of its NLS. These intermolecular spatial features may be important since the nuclear targeting motif consists mostly of basic residues that, in the cytosol, would be predicted to have a net positive charge at a pH range of ~7.40. The isoelectric point of ubiquitin is 6.79, giving ubiquitin a net negative charge under physiologic conditions. Thus, electrostatic interactions would promote ubiquitin binding to the NLS, thereby neutralizing its charge and potentially impairing the assembly of a CCTα-karyopherin cargo complex.
Nuclear import of CCTα is also essential for the generation of the nuclear membrane network (13
). A CCTα mutant devoid of a 21-residue stretch within its NH2
terminus lacked nuclear import ability and yet remained functional (30
). This NH2
-truncated CCTα mutant perhaps retains activity by relocating and binding to other membrane structures via the M domain, presumably within the cytoplasm. Still other data suggest that CCTα is stored within the nucleus when cellular requirements for PtdCho are low (24
). Upon increased demand for PtdCho synthesis, CCTα is recruited to the endoplasmic reticulum. Our model of CCTα monoubiquitination is compatible with each of these findings. When cell needs for PtdCho synthesis are low, nuclear CCTα containing an intact NLS that is disassembled by RanGTPase may be rapidly exported, monoubiquitinated, and targeted for lysosomal disposal (Fig. ). In this way, soluble, but not membrane-associated CCTα would be more prone to ubiquitination. However, in the setting of increased demand for membrane biogenesis, the levels of monoubiquitinated CCTα may be low to facilitate nuclear translocation and expansion of the nuclear reticulum (13
). Here, more CCTα would be membrane associated and relatively protected from ubiquitin modification. In support of this model, inclusion of lipid vesicles in the in vitro ubiquitination conjugation reaction or exposure of cells to oleic acid (to induce membrane binding of CCTα) reduces the pool of ubiquitinated CCTα (data not shown). Thus, perhaps CCTα membrane association and ubiquitination are linked, thereby regulating cellular PtdCho homeostasis. Last, monoubiquitinated CCTα may be modified in the cytoplasm as a mechanism to augment phospholipid synthesis. In the cytosol, sequential ubiquitin editing by actions of a deubiquitinating enzyme (DUB) could cleave ubiquitin from the CCTα-ubiquitin complex, thus preventing endosomal degradation of the enzyme (Fig. ). This scenario occurs for membrane recycling of the epithelial sodium channel (3
). Deubiquitinated or free CCTα molecules could then bind ER membranes to help catalyze the formation of PtdCho or reenter the nucleus.
FIG. 9. Model for CCTα nuclear activation, exclusion, and proteolytic processing mediated by protein monoubiquitination. A schematic diagram illustrates the proposed pathways by which CCTα is regulated by monoubiquitination. (Arrow 1) CCTα (more ...)
When analyzed by immunoblotting, considerably little CCTα is monoubiquitinated compared to the levels of deubiquitinated CCTα. The endogenous 50-kDa CCTα-ubiquitin complex is extremely difficult to detect by standard chemiluminescent analysis unless the film is exposed extensively or cells are exposed to NH4
Cl (Fig. ). These results likely reflect either very rapid translocation of CCTα-ubiquitin complexes to the lysosome coupled with efficient degradation, that monoubiquitination of CCTα occurs very slowly, or that there exists competition of substrate between ubiquitin ligases and DUBs. The latter might also explain why CCTα exhibits a relatively long t1/2
(6 to 8 h). Interestingly, the CCTK57R
mutant was retained in the nucleus and had an extremely long half-life. These results suggest that translocation of CCTα from the nucleus to the cytosol is required for its turnover and that proteasomes or proteinases within the nucleus play a lesser role in regulating enzyme stability. The translocation of CCTα might also have additional roles similar to that of other membrane proteins that require sorting into the multivesicular bodies prior to degradation in the lysosome. In this process, ubiquitin serves as a sorting signal (23