San1’s physiological function is to recognize misfolded proteins in the nucleus, targeting them for ubiquitination and proteasome degradation (Gardner et al., 2005
). San1 accomplishes substrate recognition using highly disordered N- and C-terminal arms that interact with substrates (Rosenbaum et al., 2011
). Within its substrates, San1 recognizes exposed hydrophobicity (Fredrickson et al., 2011
). Thus the means of San1 self-protection that we discovered here are consistent with San1’s function. Selective loss of Lys residues in its disordered N- and C-terminal regions provides a simple means for San1 to avoid in cis
autoubiquitination should these flexible regions come within close proximity to a bound, activated E2. Minimization of hydrophobicity in localized stretches within San1’s sequence keeps San1 from possessing the feature it recognizes in its substrates, which in turn prevents in trans
autoubiquitination of one San1 molecule by another San1 molecule.
Although the majority of the San1 “plus Lys” mutants we examined were predominantly degraded via a San1-dependent in cis mechanism, there was one peculiar class of “plus Lys” mutants whose degradation was primarily independent of San1 (N33K, N61K, and N91K). Every member of this class of mutants is within the same portion of the N-terminal region (residues 33–91), suggesting that perturbation of this region has consequences distinct from other regions in San1. However, identical to all other regions of San1, general mutation of these positions to a residue other than Lys did not lead to rapid degradation, indicating that the specific addition of a Lys residue at these positions is essential for degradation. This brings up the perplexing question as to why these particular “plus Lys” mutations undergo San1-independent degradation. One possibility is that the ubiquitination of the “plus Lys” residue specifically in this region causes structural perturbations such that another quality control E3 now targets San1. This is hard to reconcile because, if we assume the initial ubiquitination event is caused by either in cis or in trans San1-dependent ubiquitination, deletion of endogenous SAN1 and introduction of a RING-inactivating mutation should have caused complete stability of these “plus Lys” mutants. Alternatively, perhaps San1 engages another E3 (or E4) as a partner in this region, and placement of a Lys residue within this region now allows for uncontrollable ubiquitination by this partner. Future experiments are required to address how and why these specific “plus Lys” mutants are rapidly degraded via a San1-independent degradation mechanism.
The “plus Lys” San1 mutants that were subject to a primarily San1-dependent in trans
degradation mechanism (N71K, R105K, R115K) also clustered in the region immediately N-terminal to the RING domain. This region is predicted to have the highest order/structure outside of the RING domain (). It could be that these Lys mutations cause exposure of hydrophobicity that would normally be buried within a local structure, thus allowing them to be recognized as a misfolded protein by another San1 molecule. Again, this is hard to reconcile with the fact that mutation of these positions to either an Asn or Arg residue had very little effect on San1 stability (). It is therefore unlikely that the structure is perturbed by a Lys mutation. Perhaps this region is one that normally allows a functional interaction between two San1 molecules, such that placement of a Lys residue does not alter the structure but does improperly allow in trans
ubiquitination to occur. We previously explored whether one San1 molecule interacts with another, but we were unable to gather any evidence to support this possibility using either coimmunoprecipitation or two-hybrid assays (Rosenbaum et al., 2011
). It is possible that the interaction of one San1 molecule with another only occurs transiently in the context of substrate engagement to facilitate substrate polyubiquitination. If so, the highly transient San1–San1 substrate-mediated interaction would not be capable of being queried by traditional methods used to identify protein–protein interactions. Further studies will be required to reveal how and why these specific “plus Lys” San1 mutants are degraded via a San1-dependent in trans
As we noted, the in trans
degradation of the “plus hydro” San1 mutants was comparatively sluggish (). However, we could engineer the rapid degradation of the “plus hydro” San1 mutants by simply inserting a Lys residue proximal to their expanded, exposed hydrophobicity (). Previously it was found that the E3 Hrd1, which functions in the degradation of misfolded proteins in the ER (Smith et al., 2011
), uses Ser and Thr residues in addition to Lys residues for substrate ubiquitination (Ishikura et al., 2010
; Shimizu et al., 2010
). Clearly this is not the case for the “plus hydro” San1 mutants, as the endogenous Lys residues alone were essential for their degradation. We believe that our new observations have important implications for how San1 normally operates in targeting its misfolded substrates. Although we do not think that the exposed hydrophobicity in San1’s misfolded substrates must be presented in the context of disorder as appears to be the case for misfolded proteins in the endoplasmic reticulum (Xie et al., 2009
), we do now believe that the exposed hydrophobicity likely has to be proximal to Lys residues for efficient ubiquitination and rapid degradation, and no residue other than Lys can be used for San1-mediated ubiquitination. In support of this, in each San1 substrate for which we identified the regions of exposed hydrophobicity recognized by San1 (Fredrickson et al., 2011
), the exposed hydrophobic region has proximally located Lys residues (our unpublished observations), and each substrate is rapidly degraded (Fredrickson et al., 2011
Yeast possess ~50 RING E3s by homology (Li et al., 2008
). We examined this group for those that display high intrinsic disorder and contain fewer Lys residues than predicted for an average protein. Only the E3 Slx5 shared the similar characteristics seen with San1 (our unpublished observations). This is intriguing because Slx5 has also been implicated as a nuclear protein quality control E3 (Wang and Prelich, 2009
). Other protein quality control E3s in the yeast endoplasmic reticulum (Hrd1 and Doa10) or cytoplasm (Ubr1) are not highly disordered, nor do they have a below-average complement of Lys residues (our unpublished observations). Thus these sequence features might be exclusive to the nucleus, where recognition of misfolded proteins could require a different mechanism than in the endoplasmic reticulum or cytoplasm.
There are also ~300 RING E3s predicted by homology to exist in humans (Li et al., 2008
). No human E3 has been identified that functions analogously to San1 in general nuclear protein quality control degradation. One issue with identifying a human orthologue of San1 is that highly disordered proteins often have low conservation of linear sequence identity (Brown et al., 2002
), which is apparent in the fungal orthologues of San1 (). A lack of linear sequence identity conservation would confound orthologue identification searches, and so additional approaches are necessary to discover a bona fide human orthologue of San1. Because high intrinsic disorder and lack of Lys residues are conserved among San1’s fungal orthologues, we predict San1 human orthologues should also have these features. Of the ~300 human RING E3s, we identified 6 RING E3s with large regions of disorder and a reduced number of Lys residues (our unpublished observations), suggesting that these disordered E3s likely employ a similar self-protective mechanism to that of San1. It is not known whether any of these candidates functions in protein quality control degradation in the human nucleus.
Finally, we believe that our findings might also be broadly applicable to E3s that are structured. It is conceivable that some structured E3s have evolved to protect themselves by having reduced Lys residue content on their surfaces in or near the E2-binding interface or substrate-interaction regions. This idea is difficult to assess, as the large majority of solved E3/E2 structures lack the full-length E3, which means we cannot precisely know where Lys residues are located in the complete E3 tertiary structure. Although it is not clear whether Lys-residue minimization is a broader means for structured E3 self-protection, it remains a possibility to be explored as more complete structures are determined.