BACKGROUND
protein degradation via the ubiquitin proteasome system  is one of the cell's tools for selective negative regulation of intracellular proteins.
degradation via the ups has roles in maintaining protein quality control, signaling, and cell cycle progression  <cit> .
ubiquitin is a small protein that is highly conserved in eukaryotes and is the crux of the ups system.
the ups system is built upon three classes of enzymes--e <dig>  e <dig> and e3- that act sequentially to build ubiquitin chains on protein substrates.
once a protein substrate has been modified by a chain of at least four ubiquitins, it is then degraded by the 26s proteasome in an atp-dependent manner  <cit> .
the proteasome is a 33-subunit protein complex that is involved in turning over a minimum of 20% of the yeast proteome .
other lines of evidence suggest that the vast majority of cytoplasmic protein degradation is mediated by the proteasome  <cit> .
the proteasome is composed of two main components: a 20s catalytic core particle  and a 19s regulatory particle .
the 19s regulatory particle can be dissected into two sub-complexes, the lid and the base.
the base sub-complex is composed of two non-atpase subunits, rpn <dig> and rpn <dig>  as well as six atpase subunits  that are thought to unfold and feed substrate into the cp.
how ubiquitylated substrates converge onto the proteasome is an active area of research that has been studied with the greatest depth in saccharomyces cerevisiae.
so far, at least two independent mechanisms have been discovered.
in the first case, the yeast proteasome contains two intrinsic receptors, rpn <dig> and rpn <dig>  that contain defined ubiquitin binding domains  <cit> .
mammalian proteasomes contain a third intrinsic receptor, rpt <dig>  <cit> .
rpn <dig> contains a highly conserved ubiquitin interaction motif , whereas rpn <dig> binds ubiquitin via a pleckstrin motif that was not previously known to interact with ubiquitin  <cit> .
although neither rpn <dig> nor rpn <dig> is essential, rpn10Δ and rpn13Δ mutants exhibit phenotypes consistent with a role for these proteins in the docking of substrates to the proteasome.
rpt <dig> can be cross-linked to ubiquitin chains, but the means by which it binds ubiquitin and the genetic significance of this activity remain to be determined.
substrates may be able to bind the proteasome directly via these three intrinsic receptors.
in the second mode of delivery to the proteasome, receptors, including the budding yeast rad <dig>  dsk <dig>  and ddi <dig> proteins, contain an n-terminal ubiquitin like domain  that binds to the proteasome and a c- terminal ubiquitin association domain  that binds to ubiquitin chains  <cit> .
unlike rpn <dig> and rpn <dig>  these proteins are not stoichiometric subunits of the proteasome.
instead, it is thought that this class of proteins rapidly cycles on and off the proteasome  <cit> , serving as 'shuttle' receptors that bind substrates in the cytoplasm and nucleus and deliver them to the proteasome.
the uba-ubl proteins dock at the proteasome by binding the largest subunit of the proteasome, rpn <dig>  <cit> , although recent evidence suggests that the uba-ubl proteins also bind other subunits within the proteasome.
for example, multiple lines of evidence suggest that in yeast dsk <dig> may also be able to interact with rpn <dig> and rpn <dig>  and yeast rad <dig> may also bind rpt <dig>  <cit> .
human rad <dig> is also able to bind both human rpn <dig> and rpn <dig>  <cit>  and in an nmr experiment, binding of yeast rad <dig> to rpn <dig> was observed  <cit> .
while it is clear that substrates can use two different mechanisms to engage the proteasome, we still do not understand how substrates are allocated to one targeting pathway or the other.
while there is evidence that some protein substrates utilize both the intrinsic and shuttling receptors  <cit> , some proteasomal substrates are entirely dependent on either rpn <dig> or rad <dig>  <cit> .
moreover, although rpn <dig> and rpn <dig> are undoubtedly important receptors, electron microscopy and quantitative mass spectrometry data suggest that there are two populations of proteasomes, those containing and those not containing the intrinsic receptors  <cit> .
furthermore, deletion of rpn <dig> or rpn <dig> does not lead to profound deficits in cellular protein degradation  <cit> .
finally, while highly conserved  <cit> , the uba-ubl proteins are not essential for yeast cell growth  <cit> .
thus, although the proteasome itself is essential, none of the receptors that link substrates to the proteasome  is essential.
this has led to the assumption that targeting of substrates to the proteasome occurs by multiple, partially redundant mechanisms.
obtaining a clear understanding of how each pathway contributes to substrate recognition by the proteasome is of considerable importance given the central role of the ups in regulatory biology and the clinical significance of the proteasome as a target for cancer therapy  <cit> .
rpn <dig>  the largest subunit of the proteasome, contains nine repeat segments, known as leucine rich repeats , which adopt horseshoe-shaped structures that are thought to be generally important for protein-protein interactions  <cit> .
the lrr domain of rpn <dig> is thought to form a slightly open monomeric α-solenoid  <cit> .
the first five contiguous repeat segments constitute lrr <dig>  whereas the next four contiguous lrr repeats form lrr <dig>  a  <dig> acidic amino acid stretch links lrr <dig> and lrr <dig>  <cit> .
the minimal region sufficient for rad <dig> binding to rpn <dig> has been mapped to residues  <dig> to  <dig>  which comprise lrr <dig> and an adjacent 21-residue acidic stretch on the c-terminal side.
the ubl domains of dsk <dig> and ddi <dig> have also been shown to interact with the lrr domain of rpn <dig>  <cit> .
to gain a better understanding of how substrate delivery to the proteasome is controlled, we sought to identify an rpn <dig> mutant that is defective in binding the uba-ubl receptor proteins.
we identified two mutations that disrupted binding of the uba-ubl protein ddi <dig> to the proteasome.
docking of dsk <dig> to the proteasome was also moderately affected by these mutations in some genetic backgrounds.
the delivery of ubiquitin conjugates to the proteasome was diminished in an rpn1-d517a single and even more so in an rpn13Δ rpn1-d517a double mutant.
lastly, we show that the rpn1-d517a mutation stabilizes the ddi <dig> substrate, ufo <dig>
RESULTS
rpn <dig> to  <dig> interacts with ubl domain proteins
to screen for mutations in rpn <dig> that disrupt binding to uba-ubl proteins, we engineered a reverse yeast two-hybrid system that reports on association between a fragment of rpn <dig> , a fragment including regions previously shown to be necessary and sufficient for uba-ubl binding and four distinct ubl-containing proteins  known to interact with the proteasome  <cit> .
productive binding between rpn <dig> to  <dig> and ubl proteins was expected to drive expression of his <dig> and ura <dig>  resulting in growth on 3-aminotriazole  and inability to grow on 5-fluoroorotic acid  .
growth assays revealed that rpn <dig> to  <dig> was capable of binding to rad <dig>  dsk <dig>  ddi <dig> and ubp <dig> in yeast cells, whereas a ddi <dig> fragment lacking its ubl domain and rpn <dig>  a proteasomal subunit, were unable to bind rpn <dig> to  <dig> .
identification of mutations in rpn <dig> to  <dig> that block binding to ubl domain proteins
using growth on 5foa as a positive selection for loss of interaction between rpn <dig> to  <dig> and ubl proteins, we screened a pcr-mutagenized allele library containing over  <dig>  individual clones coding for rpn <dig> to  <dig> and selected for mutants that could no longer interact with rad <dig>  or dsk <dig> .
we screened these  <dig>  transformants for their ability to reproduce their 5foar phenotype.
one hundred, ninety colonies that again tested 5foar were sequenced.
forty-two of the sequenced 5foar clones contained a mutation; single amino acid substitutions were identified in  <dig> clones, while silent mutations , and truncation or frameshift events  made up the remainder.
plasmids containing single rpn <dig> mutations were then retransformed and assayed for their ability to reproduce the 5foar phenotype.
twelve amino acid substitutions in  <dig> different residues of rpn <dig> were identified as testing positive after being retransformed into our ry2h reporter strain .
given that a structural model of the lrr region of rpn <dig> exists  <cit> , we also generated a panel of six 'rational' mutations that perturb residues predicted to be on the outside surface of the lrr domain .
the relative positions of the mutations discovered in the 'reverse yeast 2-hybrid' screen and the rational mutations are shown on the model structure of the rpn <dig> lrr domain .
mutant rpn <dig> alleles display synthetic growth defects in combination with ubiquitin receptor mutants
to evaluate whether any of the mutations in our panel of  <dig> substitutions had an effect on proteasome function, we reconstructed them into full-length rpn <dig> and performed a 'plasmid shuffle' to replace the essential rpn <dig> gene with each of our mutant alleles.
a yeast rpn1Δ strain sustained by wild type rpn <dig> on a ura <dig> plasmid was individually transformed with a leu <dig> plasmid bearing each mutant rpn <dig> allele and the cells were plated on 5foa to identify clones from which the ura <dig> plasmid was evicted.
we recovered 5foa-resistant colonies from all transformants with the exception of rpn1f534s, indicating that  <dig> of our alleles retained at least partial rpn <dig> function.
to evaluate the impact of our rpn <dig> mutations on proteasome function, we plated cells on medium supplemented with the proline analog l-azetidine-2-carboxylic acid .
cells with defective proteasome function are sensitive to azc  <cit> , presumably because its incorporation into proteins causes misfolding, thereby placing an elevated demand on cellular quality-control pathways.
as shown in figure 2a and additional file  <dig>  figure s1a, none of our mutants was hyper-sensitive to azc.
multiple receptors dock ubiquitinated substrates to the proteasome, including not only the ubl domain proteins but also rpn <dig> and rpn <dig>  <cit> .
unlike the other subunits of the proteasome, rpn <dig> and rpn <dig> are not essential.
therefore, we sought to test whether mutations in these receptors might sensitize cells to our rpn <dig> alleles.
deletion of rpn <dig> by itself did not cause sensitivity to azc .
however, a subset of our rpn <dig> mutants  exhibited striking sensitivity to azc when combined with rpn13Δ .
to test whether this synthetic defect was due to the role of rpn <dig> as an ub receptor, the five rpn <dig> mutants showing the most striking phenotypes were introduced by plasmid shuffle into an rpn13-kkd strain that contains a triple point mutation that inactivates the ubiquitin binding domain  <cit> .
four of the five tested rpn <dig> alleles showed a similar synthetic growth defect in the rpn13-kkd mutant background .
these data indicate that our rpn <dig> mutant proteins sensitized cells to loss of an intrinsic proteasome ubiquitin receptor.
given the synthetic effects seen with rpn <dig> alleles, we sought to test whether the same subset of rpn <dig> mutants exhibited genetic interaction with rpn <dig>  rpn <dig> contains two domains: a vwa domain that appears to play a structural role and an ubiquitin-binding uim domain.
we used plasmid shuffle to introduce rpn <dig> alleles into a mutant, rpn10-uim, in which the uim domain is inactivated by a cluster of point mutations  <cit> .
whereas neither the individual rpn <dig> mutants  nor rpn10-uim  was hypersensitive to azc, the double mutants exhibited striking sensitivity .
similarly, we also found that the same rpn <dig> alleles further sensitized an rpn13-kkd rpn10-uim strain to azc .
as a test for specificity, we introduced the same set of rpn <dig> mutations into an rpn4Δ background.
rpn <dig> is a transcription factor that promotes proteasome gene expression, and rpn4Δ mutants have reduced proteasome levels and show synthetic phenotypes with a number of mutations that impinge on proteasome function  <cit> .
in contrast to the results seen with rpn13Δ, rpn13-kkd, and rpn10-uim, none of the five rpn <dig> mutants tested exhibited a synthetic azc-sensitive phenotype when combined with rpn4Δ .
taken together, these data suggest that the rpn <dig> mutant alleles impinge specifically on compromised receptor function, and do not cause general proteasome impairment.
recruitment of ddi <dig>  dsk <dig> and ubiquitin conjugates to proteasomes is compromised in rpn1-d517a and rpn1-k484a mutants
we next aimed to determine if any of the rpn <dig> mutations that showed genetic interactions with rpn10-uim and rpn13-kkd led to defects in recruitment of ubl containing proteins to the proteasome.
to address this question, we first tagged rpn <dig> with sequences encoding the flag epitope in a selection of rpn13Δ rpn <dig> mutants.
we included rpn13Δ in this analysis due to potential redundancy between rpn <dig> and rpn <dig> for binding ubl domains.
proteasomes were immunoprecipitated from these strains and immunoblotted for the presence of ubl proteins.
all double mutant proteasomes that were analyzed contained equivalent levels of associated rad <dig>  dsk <dig> and rpn <dig> except for rpn1-d517a and rpn1-k484a, both of which exhibited reduced levels of bound dsk <dig> .
none of our rpn <dig> single mutants by themselves or in the rpn10-uim background showed significantly reduced levels of proteasome-bound dsk <dig> .
to see if we could identify other binding-defective rpn <dig> mutations, we generated an additional set of 'rational' rpn <dig> alleles and tested them by using plasmid shuffle to introduce the alleles into an rpn11flag rpn1Δ rpn13Δ background, followed by immunoprecipitation of the proteasomes and immunoblotting for ubl proteins.
none of these mutants, which are listed in additional file  <dig>  table s <dig>  exhibited a greater ubl binding defect than the d517a or k484a alleles and so they were not pursued further.
based on the proteasome association studies, we focused our attention on the k484a and d517a mutants.
to evaluate the association of ubl proteins in greater depth, we retrieved proteasomes from both mutants  and immunoblotted the immunoprecipitates to determine their content of rad <dig>  dsk <dig>  ddi <dig>  ubp <dig>  and total ubiquitin conjugates.
the immunoblots are shown in the left panels of figure 3b and densitometric quantification of the results is presented in the right panel.
proteasomes from both rpn <dig> rpn13Δ mutants contained normal or near-normal levels of the ubl proteins ubp <dig> and rad <dig>  in this experiment the levels of dsk <dig> were higher than those observed in figure 3a, possibly because the immunoprecipitation was done under less stringent conditions.
interestingly, proteasomes recovered from rpn13Δ rpn1-d517a cells contained reduced levels of ddi <dig> and total ubiquitin conjugates compared to proteasomes retrieved from either wild type or rpn13Δ rpn1-k484a cells.
similar results were obtained with proteasomes from rpn1-d517a and rpn10-uim rpn1-d517a cells .
).
these results indicate that mutation of the d <dig> residue of rpn <dig> by itself was sufficient to destabilize ddi <dig> docking, and in combination with loss of rpn <dig> modestly destabilized dsk <dig> binding.
we were intrigued by the mild defect in dsk <dig> binding to rpn13Δrpn1-d517a proteasomes and questioned if combining rpn10-uim, rpn13-kkd, and rpn1-d517a mutations might yield a stronger defect in recruitment of dsk <dig> since interaction of the ubl proteins has been observed with rpn <dig> and rpn <dig>  <cit> .
we retrieved  <dig> proteasomes from both a double rpn10-uim rpn13-kkd and a triple rpn10-uim rpn13- kkd rpn1-d517a mutant and immunoblotted the immunoprecipitates to determine their content of dsk <dig> and ddi <dig>  proteasomes from an rpn10-uim rpn13-kkd rpn1-d517a strain contained fewer ub conjugates in comparison to an isogenic strain containing wild type rpn <dig> .
additionally, we quantified the change in the binding of ddi <dig> and dsk <dig> and again observed diminished recruitment of both proteins in the presence of the rpn1-d517a mutation .
this led us to investigate the effect of the rpn1-d517a mutation in greater detail.
to determine whether rpn1-d517a proteasomes were generally defective, we characterized them biochemically and found them to be completely normal by multiple methods.
purified rpn1-d517a proteasomes exhibited a normal subunit composition when evaluated by sds-page .
moreover, native nondenaturing gel electrophoresis verified that these proteasomes  and those of all of the other strains indicated in figure 1d  were properly assembled and had normal chymotryptic activity.
in fact, although we were manipulating the largest scaffolding subunit of the proteasome, only a small number of the mutations we studied had any negative consequences on proteasome stability .
to characterize in detail the impact of the rpn1-d517a mutation on proteasome composition, we performed a quantitative mass spectrometry technique, silac .
for this experiment,rpn13Δ cells were grown in medium supplemented with heavy isotopes of lysine and arginine while rpn13Δ rpn1-d517a cells were grown in medium with 'light' lysine and arginine.
the two cultures were mixed immediately prior to lysis and proteasomes were purified by affinity chromatography on an anti-flag resin.
the purified sample was then analyzed by multidimensional mass spectrometry and the heavy/light ratios for peptides derived from proteasome subunits were determined .
this sensitive analysis confirmed that rpn1-d517a does not cause any apparent physical change in the proteasome.
while we did measure a slightly reduced level of rad <dig> in rpn13Δ rpn1-d517a proteasomes by immunoblotting, our silac data indicated that the levels of rad <dig> and ubp <dig> were largely unaffected in rpn13Δ rpn1-d517a proteasomes, as they had heavy to light  ratios of  <dig>  and  <dig>  respectively.
this is not unexpected, since it was reported in a prior silac study that the free and proteasome-bound pools of human rad <dig> rapidly equilibrate in cell lysate  <cit> .
unfortunately, ddi <dig> and dsk <dig> peptides were not seen in our silac experiment.
capturing the association of all three uba-ubl receptor proteins with proteasomes in native preparations is challenging, likely because these proteins interact very dynamically with the proteasome  <cit> .
for instance, the association of dsk <dig> with the proteasome has been reported to be difficult to capture  <cit> .
additionally, only one published mass spectrometry study has been able to simultaneously capture rad <dig>  dsk <dig> and ddi <dig> with the proteasome, and that study relied on chemical cross-linking to stabilize the association of dynamically-bound proteasome interactors  <cit> .
in vitro confirmation of a ddi <dig> binding defect of rpn13-deficient rpn1-d517a mutant proteasomes
as an orthogonal approach, we sought to perform an in vitro binding assay that would confirm our analysis of proteasomes purified from mutant cells.
proteasomes were affinity purified from cells expressing flag-tagged rpn <dig> and incubated with recombinant gst-uba-ubl proteins.
proteasomes affinity purified from rpn13Δ cells were successfully pulled-down by all three baits.
however, rpn13-deficient rpn1-v447h k484a d517a  proteasomes exhibited strongly diminished binding capacity for ddi <dig> .
it should be noted that rpn13-deficient rpn1-v447h k484a d517a proteasome mutants behaved just as a rpn13-deficient rpn1-d517a mutant proteasomes in native immunoprecipitation experiments .
however, it was surprising that we did not see a loss of dsk <dig> binding to proteasomes isolated from an rpn13Δrpn1-vkd strain.
it is possible that the effect of the d517a mutation on dsk <dig> binding was subtle, and was overcome by mass action due to the high level of gst-dsk <dig> employed in the pull-down.
rpn1-d517a mutants exhibit a selective defect in protein degradation
it is thought that uba-ubl proteins exhibit some degree of selectivity in targeting specific substrates to the proteasome  <cit> .
we hypothesized that the decrease of ddi <dig> binding to proteasomes in an rpn1-d517a mutant might therefore result in turnover defects of substrates that are particularly reliant on ddi <dig>  in agreement with the normal binding of rad <dig> and dsk <dig> to the proteasome in an rpn1-d517a single mutant, no defect was seen in turnover of the rad23/dsk2-dependent substrate, cpy*   <cit> , or the dsk <dig> substrate kre <dig>   <cit> .
however, when we tested the ddi1-dependent substrate, ufo <dig>  <cit> , we saw nearly complete stabilization in comparison to a wild type strain .
note that the initial levels of plasmid encoded gst-ufo <dig> were higher in rpn1-d517a compared to ddi1Δ cells.
this was seen in two of three replicates.
we do not know the basis for this.
in agreement with the turnover data, we observed that over-expression of gst-ufo <dig> was toxic to ddi1Δ and rpn1-d517a cells but not wild type cells .
this effect was exquisitely specific--neither rpn13Δ  nor any other mutation in rpn <dig> that we tested  conferred sensitivity to over-expression of dst-ufo <dig>
discussion
of the three uba-ubl shuttle receptors linked to the proteasome, ddi <dig> is the least studied and perhaps the most controversial.
prior data have established that ddi <dig> binds polyubiquitin, albeit with lower affinity than rad <dig> and dsk <dig>  <cit> .
however, while some studies report a physical interaction of ddi <dig> with rpn <dig> or the intact proteasome  <cit> , there are a few reports that question the capacity for ddi <dig> to bind the proteasome or rpn <dig>  <cit> .
the disparity in these reports may be due to the qualitative nature of immunoprecipitation experiments and the rapid dynamics of ubl binding to and dissociation from the proteasome  <cit> .
ddi <dig> has the most divergent ubl domain among the known uba-ubl proteins, and hence, may have the weakest affinity interaction with the proteasome  <cit> .
we have shown that ddi <dig> is recovered with proteasomes immunoprecipitated from yeast cells, binds rpn <dig> in a yeast two-hybrid assay, and binds to the proteasome in an in vitro pull-down assay.
we have further validated these results by identifying an rpn <dig> mutation that is selectively defective in binding ddi <dig> and stabilizes the ddi1-dependent proteasome substrate ufo <dig>  hence, we conclude that ddi <dig> does indeed interact with the proteasome in a specific and functionally-relevant manner.
if ddi <dig> binds the proteasome more weakly than other uba-ubl proteins, which seems likely, it could explain why the d517a and k484a mutations reported here selectively disrupt interaction of ddi <dig> with the proteasome.
our study highlights the layered complexity of the interaction of shuttle proteins with the proteasome.
with a single alanine substitution in the highly conserved d <dig> residue of rpn <dig> we were able to significantly reduce the binding of ddi <dig> to the proteasome.
however, the interaction of other uba-ubl proteins with the proteasome appears to be more complex.
recovery of dsk <dig> with proteasomes was only mildly diminished in an rpn1-d517a mutant that also lacked rpn <dig> or the ubiquitin interaction motifs of both rpn <dig> and rpn <dig>  meanwhile, recovery of rad <dig> was not affected appreciably by any mutation in rpn <dig> analyzed during the course of this work.
there are two possible explanations of these results.
on the one hand, it is possible that the domains of these proteins have a gradient of affinity for rpn <dig>  with ddi <dig> being the weakest binder and rad <dig> the strongest.
in this scenario, rpn1-d517a may be a hypomorph that only modestly perturbs the ubl docking site, such that only the weakest binder  is excluded.
we attempted to test this hypothesis by making numerous combinatorial mutations, , none of which exhibited a substantially greater ubl binding defect than the d517a or k484a alleles .
thus, we do not favor the hypothesis that it is possible to disrupt recruitment of rad <dig> and dsk <dig> by mutating a single binding patch on rpn <dig>
on the other hand, our model for ubl docking to the proteasome suggests that it is possible that ddi <dig> uses only a single mechanism to bind the proteasome , whereas, in line with published reports  <cit> , rad <dig> and dsk <dig> may use multiple mechanisms  and thus are more resistant to mutation .
our observation that reduction of dsk <dig> binding was only seen in an rpn1-d517a rpn13Δ double mutant and more strikingly in a rpn1-d517a rpn13Δ rpn10-uim supports the idea that dsk <dig> may be tethered to the proteasome by either rpn <dig>  rpn <dig> or rpn <dig> .
the failure to see a significant reduction in binding of rad <dig> in any single or double mutant may be due to there being multiple independent docking sites for rad <dig> on the proteasome, although it should be noted that all of these docking sites appear to rely on the ubl domain  <cit> .
other studies have shown that ubp <dig> may bind proteasome lid proteins while rad <dig> may also bind rpt <dig>  <cit> , and that even ub chains bound to rad <dig> may contribute to its proteasome binding  <cit> .
biochemical data suggest that human rad <dig> is recruited to the proteasome by the uim domain of rpn <dig>  <cit> , and that even yeast rad <dig> can bind rpn <dig>  <cit>  but it should be noted that this hypothesis has not been tested by genetic manipulation of rpn <dig> in cells.
clearly, more work is needed to unravel the mechanisms underlying recruitment of the ubl domain proteins of rad <dig>  dsk <dig>  and ubp <dig> to the proteasome.
CONCLUSIONS
the current study identifies residues in the lrr <dig> domain of rpn <dig> that contribute to shuttle receptor docking.
we validate ddi <dig> as a proteasomal shuttle receptor whose stable binding to the proteasome depends on rpn <dig> residue d <dig>  consistent with this, d <dig> is also important for the degradation of a ddi1-dependent substrate.
we also show that in the absence of rpn <dig>  or the dual absence of the ubiquitin binding domains of rpn <dig> and rpn <dig>  mutation of the d <dig> or k <dig> residues reduces the association of dsk <dig> with the proteasome.

 protein degradation via the ubiquitin proteasome system is one of the cell's tools for selective negative regulation of intracellular proteins. degradation via the ups has roles in maintaining protein quality control, signaling, and cell cycle progression. ubiquitin is a small protein that is highly conserved in eukaryotes and is the crux of the ups system. the ups system is built upon three classes of enzymes--e e and e3- that act sequentially to build ubiquitin chains on protein substrates. once a protein substrate has been modified by a chain of at least four ubiquitins, it is then degraded by the 26s proteasome in an atp-dependent manner. the proteasome is a 33-subunit protein complex that is involved in turning over a minimum of 20% of the yeast proteome. other lines of evidence suggest that the vast majority of cytoplasmic protein degradation is mediated by the proteasome. the proteasome is composed of two main components: a 20s catalytic core particle and a 19s regulatory particle. the 19s regulatory particle can be dissected into two sub-complexes, the lid and the base. the base sub-complex is composed of two non-atpase subunits, rpn and rpn as well as six atpase subunits that are thought to unfold and feed substrate into the cp. how ubiquitylated substrates converge onto the proteasome is an active area of research that has been studied with the greatest depth in saccharomyces cerevisiae. so far, at least two independent mechanisms have been discovered. in the first case, the yeast proteasome contains two intrinsic receptors, rpn and rpn that contain defined ubiquitin binding domains. mammalian proteasomes contain a third intrinsic receptor, rpt. rpn contains a highly conserved ubiquitin interaction motif, whereas rpn binds ubiquitin via a pleckstrin motif that was not previously known to interact with ubiquitin. although neither rpn nor rpn is essential, rpn10Δ and rpn13Δ mutants exhibit phenotypes consistent with a role for these proteins in the docking of substrates to the proteasome. rpt can be cross-linked to ubiquitin chains, but the means by which it binds ubiquitin and the genetic significance of this activity remain to be determined. substrates may be able to bind the proteasome directly via these three intrinsic receptors. in the second mode of delivery to the proteasome, receptors, including the budding yeast rad dsk and ddi proteins, contain an n-terminal ubiquitin like domain that binds to the proteasome and a c- terminal ubiquitin association domain that binds to ubiquitin chains. unlike rpn and rpn these proteins are not stoichiometric subunits of the proteasome. instead, it is thought that this class of proteins rapidly cycles on and off the proteasome, serving as'shuttle' receptors that bind substrates in the cytoplasm and nucleus and deliver them to the proteasome. the uba-ubl proteins dock at the proteasome by binding the largest subunit of the proteasome, rpn, although recent evidence suggests that the uba-ubl proteins also bind other subunits within the proteasome. for example, multiple lines of evidence suggest that in yeast dsk may also be able to interact with rpn and rpn and yeast rad may also bind rpt. human rad is also able to bind both human rpn and rpn and in an nmr experiment, binding of yeast rad to rpn was observed. while it is clear that substrates can use two different mechanisms to engage the proteasome, we still do not understand how substrates are allocated to one targeting pathway or the other. while there is evidence that some protein substrates utilize both the intrinsic and shuttling receptors, some proteasomal substrates are entirely dependent on either rpn or rad. moreover, although rpn and rpn are undoubtedly important receptors, electron microscopy and quantitative mass spectrometry data suggest that there are two populations of proteasomes, those containing and those not containing the intrinsic receptors. furthermore, deletion of rpn or rpn does not lead to profound deficits in cellular protein degradation. finally, while highly conserved, the uba-ubl proteins are not essential for yeast cell growth. thus, although the proteasome itself is essential, none of the receptors that link substrates to the proteasome is essential. this has led to the assumption that targeting of substrates to the proteasome occurs by multiple, partially redundant mechanisms. obtaining a clear understanding of how each pathway contributes to substrate recognition by the proteasome is of considerable importance given the central role of the ups in regulatory biology and the clinical significance of the proteasome as a target for cancer therapy. rpn the largest subunit of the proteasome, contains nine repeat segments, known as leucine rich repeats, which adopt horseshoe-shaped structures that are thought to be generally important for protein-protein interactions. the lrr domain of rpn is thought to form a slightly open monomeric α-solenoid. the first five contiguous repeat segments constitute lrr whereas the next four contiguous lrr repeats form lrr a acidic amino acid stretch links lrr and lrr. the minimal region sufficient for rad binding to rpn has been mapped to residues to which comprise lrr and an adjacent 21-residue acidic stretch on the c-terminal side. the ubl domains of dsk and ddi have also been shown to interact with the lrr domain of rpn. to gain a better understanding of how substrate delivery to the proteasome is controlled, we sought to identify an rpn mutant that is defective in binding the uba-ubl receptor proteins. we identified two mutations that disrupted binding of the uba-ubl protein ddi to the proteasome. docking of dsk to the proteasome was also moderately affected by these mutations in some genetic backgrounds. the delivery of ubiquitin conjugates to the proteasome was diminished in an rpn1-d517a single and even more so in an rpn13Δ rpn1-d517a double mutant. lastly, we show that the rpn1-d517a mutation stabilizes the ddi substrate, ufo  rpn to interacts with ubl domain proteins to screen for mutations in rpn that disrupt binding to uba-ubl proteins, we engineered a reverse yeast two-hybrid system that reports on association between a fragment of rpn, a fragment including regions previously shown to be necessary and sufficient for uba-ubl binding and four distinct ubl-containing proteins known to interact with the proteasome. productive binding between rpn to and ubl proteins was expected to drive expression of his and ura resulting in growth on 3-aminotriazole and inability to grow on 5-fluoroorotic acid. growth assays revealed that rpn to was capable of binding to rad dsk ddi and ubp in yeast cells, whereas a ddi fragment lacking its ubl domain and rpn a proteasomal subunit, were unable to bind rpn to. identification of mutations in rpn to that block binding to ubl domain proteins using growth on 5foa as a positive selection for loss of interaction between rpn to and ubl proteins, we screened a pcr-mutagenized allele library containing over individual clones coding for rpn to and selected for mutants that could no longer interact with rad or dsk. we screened these transformants for their ability to reproduce their 5foar phenotype. one hundred, ninety colonies that again tested 5foar were sequenced. forty-two of the sequenced 5foar clones contained a mutation; single amino acid substitutions were identified in clones, while silent mutations, and truncation or frameshift events made up the remainder. plasmids containing single rpn mutations were then retransformed and assayed for their ability to reproduce the 5foar phenotype. twelve amino acid substitutions in different residues of rpn were identified as testing positive after being retransformed into our ry2h reporter strain. given that a structural model of the lrr region of rpn exists, we also generated a panel of six'rational' mutations that perturb residues predicted to be on the outside surface of the lrr domain. the relative positions of the mutations discovered in the'reverse yeast 2-hybrid' screen and the rational mutations are shown on the model structure of the rpn lrr domain. mutant rpn alleles display synthetic growth defects in combination with ubiquitin receptor mutants to evaluate whether any of the mutations in our panel of substitutions had an effect on proteasome function, we reconstructed them into full-length rpn and performed a'plasmid shuffle' to replace the essential rpn gene with each of our mutant alleles. a yeast rpn1Δ strain sustained by wild type rpn on a ura plasmid was individually transformed with a leu plasmid bearing each mutant rpn allele and the cells were plated on 5foa to identify clones from which the ura plasmid was evicted. we recovered 5foa-resistant colonies from all transformants with the exception of rpn1f534s, indicating that of our alleles retained at least partial rpn function. to evaluate the impact of our rpn mutations on proteasome function, we plated cells on medium supplemented with the proline analog l-azetidine-2-carboxylic acid. cells with defective proteasome function are sensitive to azc, presumably because its incorporation into proteins causes misfolding, thereby placing an elevated demand on cellular quality-control pathways. as shown in figure 2a and additional file figure s1a, none of our mutants was hyper-sensitive to azc. multiple receptors dock ubiquitinated substrates to the proteasome, including not only the ubl domain proteins but also rpn and rpn. unlike the other subunits of the proteasome, rpn and rpn are not essential. therefore, we sought to test whether mutations in these receptors might sensitize cells to our rpn alleles. deletion of rpn by itself did not cause sensitivity to azc. however, a subset of our rpn mutants exhibited striking sensitivity to azc when combined with rpn13Δ. to test whether this synthetic defect was due to the role of rpn as an ub receptor, the five rpn mutants showing the most striking phenotypes were introduced by plasmid shuffle into an rpn13-kkd strain that contains a triple point mutation that inactivates the ubiquitin binding domain. four of the five tested rpn alleles showed a similar synthetic growth defect in the rpn13-kkd mutant background. these data indicate that our rpn mutant proteins sensitized cells to loss of an intrinsic proteasome ubiquitin receptor. given the synthetic effects seen with rpn alleles, we sought to test whether the same subset of rpn mutants exhibited genetic interaction with rpn rpn contains two domains: a vwa domain that appears to play a structural role and an ubiquitin-binding uim domain. we used plasmid shuffle to introduce rpn alleles into a mutant, rpn10-uim, in which the uim domain is inactivated by a cluster of point mutations. whereas neither the individual rpn mutants nor rpn10-uim was hypersensitive to azc, the double mutants exhibited striking sensitivity. similarly, we also found that the same rpn alleles further sensitized an rpn13-kkd rpn10-uim strain to azc. as a test for specificity, we introduced the same set of rpn mutations into an rpn4Δ background. rpn is a transcription factor that promotes proteasome gene expression, and rpn4Δ mutants have reduced proteasome levels and show synthetic phenotypes with a number of mutations that impinge on proteasome function. in contrast to the results seen with rpn13Δ, rpn13-kkd, and rpn10-uim, none of the five rpn mutants tested exhibited a synthetic azc-sensitive phenotype when combined with rpn4Δ. taken together, these data suggest that the rpn mutant alleles impinge specifically on compromised receptor function, and do not cause general proteasome impairment. recruitment of ddi dsk and ubiquitin conjugates to proteasomes is compromised in rpn1-d517a and rpn1-k484a mutants we next aimed to determine if any of the rpn mutations that showed genetic interactions with rpn10-uim and rpn13-kkd led to defects in recruitment of ubl containing proteins to the proteasome. to address this question, we first tagged rpn with sequences encoding the flag epitope in a selection of rpn13Δ rpn mutants. we included rpn13Δ in this analysis due to potential redundancy between rpn and rpn for binding ubl domains. proteasomes were immunoprecipitated from these strains and immunoblotted for the presence of ubl proteins. all double mutant proteasomes that were analyzed contained equivalent levels of associated rad dsk and rpn except for rpn1-d517a and rpn1-k484a, both of which exhibited reduced levels of bound dsk. none of our rpn single mutants by themselves or in the rpn10-uim background showed significantly reduced levels of proteasome-bound dsk. to see if we could identify other binding-defective rpn mutations, we generated an additional set of'rational' rpn alleles and tested them by using plasmid shuffle to introduce the alleles into an rpn11flag rpn1Δ rpn13Δ background, followed by immunoprecipitation of the proteasomes and immunoblotting for ubl proteins. none of these mutants, which are listed in additional file table s exhibited a greater ubl binding defect than the d517a or k484a alleles and so they were not pursued further. based on the proteasome association studies, we focused our attention on the k484a and d517a mutants. to evaluate the association of ubl proteins in greater depth, we retrieved proteasomes from both mutants and immunoblotted the immunoprecipitates to determine their content of rad dsk ddi ubp and total ubiquitin conjugates. the immunoblots are shown in the left panels of figure 3b and densitometric quantification of the results is presented in the right panel. proteasomes from both rpn rpn13Δ mutants contained normal or near-normal levels of the ubl proteins ubp and rad in this experiment the levels of dsk were higher than those observed in figure 3a, possibly because the immunoprecipitation was done under less stringent conditions. interestingly, proteasomes recovered from rpn13Δ rpn1-d517a cells contained reduced levels of ddi and total ubiquitin conjugates compared to proteasomes retrieved from either wild type or rpn13Δ rpn1-k484a cells. similar results were obtained with proteasomes from rpn1-d517a and rpn10-uim rpn1-d517a cells. ). these results indicate that mutation of the d residue of rpn by itself was sufficient to destabilize ddi docking, and in combination with loss of rpn modestly destabilized dsk binding. we were intrigued by the mild defect in dsk binding to rpn13Δrpn1-d517a proteasomes and questioned if combining rpn10-uim, rpn13-kkd, and rpn1-d517a mutations might yield a stronger defect in recruitment of dsk since interaction of the ubl proteins has been observed with rpn and rpn. we retrieved proteasomes from both a double rpn10-uim rpn13-kkd and a triple rpn10-uim rpn13- kkd rpn1-d517a mutant and immunoblotted the immunoprecipitates to determine their content of dsk and ddi proteasomes from an rpn10-uim rpn13-kkd rpn1-d517a strain contained fewer ub conjugates in comparison to an isogenic strain containing wild type rpn. additionally, we quantified the change in the binding of ddi and dsk and again observed diminished recruitment of both proteins in the presence of the rpn1-d517a mutation. this led us to investigate the effect of the rpn1-d517a mutation in greater detail. to determine whether rpn1-d517a proteasomes were generally defective, we characterized them biochemically and found them to be completely normal by multiple methods. purified rpn1-d517a proteasomes exhibited a normal subunit composition when evaluated by sds-page. moreover, native nondenaturing gel electrophoresis verified that these proteasomes and those of all of the other strains indicated in figure 1d were properly assembled and had normal chymotryptic activity. in fact, although we were manipulating the largest scaffolding subunit of the proteasome, only a small number of the mutations we studied had any negative consequences on proteasome stability. to characterize in detail the impact of the rpn1-d517a mutation on proteasome composition, we performed a quantitative mass spectrometry technique, silac. for this experiment,rpn13Δ cells were grown in medium supplemented with heavy isotopes of lysine and arginine while rpn13Δ rpn1-d517a cells were grown in medium with'light' lysine and arginine. the two cultures were mixed immediately prior to lysis and proteasomes were purified by affinity chromatography on an anti-flag resin. the purified sample was then analyzed by multidimensional mass spectrometry and the heavy/light ratios for peptides derived from proteasome subunits were determined. this sensitive analysis confirmed that rpn1-d517a does not cause any apparent physical change in the proteasome. while we did measure a slightly reduced level of rad in rpn13Δ rpn1-d517a proteasomes by immunoblotting, our silac data indicated that the levels of rad and ubp were largely unaffected in rpn13Δ rpn1-d517a proteasomes, as they had heavy to light ratios of and respectively. this is not unexpected, since it was reported in a prior silac study that the free and proteasome-bound pools of human rad rapidly equilibrate in cell lysate. unfortunately, ddi and dsk peptides were not seen in our silac experiment. capturing the association of all three uba-ubl receptor proteins with proteasomes in native preparations is challenging, likely because these proteins interact very dynamically with the proteasome. for instance, the association of dsk with the proteasome has been reported to be difficult to capture. additionally, only one published mass spectrometry study has been able to simultaneously capture rad dsk and ddi with the proteasome, and that study relied on chemical cross-linking to stabilize the association of dynamically-bound proteasome interactors. in vitro confirmation of a ddi binding defect of rpn13-deficient rpn1-d517a mutant proteasomes as an orthogonal approach, we sought to perform an in vitro binding assay that would confirm our analysis of proteasomes purified from mutant cells. proteasomes were affinity purified from cells expressing flag-tagged rpn and incubated with recombinant gst-uba-ubl proteins. proteasomes affinity purified from rpn13Δ cells were successfully pulled-down by all three baits. however, rpn13-deficient rpn1-v447h k484a d517a proteasomes exhibited strongly diminished binding capacity for ddi. it should be noted that rpn13-deficient rpn1-v447h k484a d517a proteasome mutants behaved just as a rpn13-deficient rpn1-d517a mutant proteasomes in native immunoprecipitation experiments. however, it was surprising that we did not see a loss of dsk binding to proteasomes isolated from an rpn13Δrpn1-vkd strain. it is possible that the effect of the d517a mutation on dsk binding was subtle, and was overcome by mass action due to the high level of gst-dsk employed in the pull-down. rpn1-d517a mutants exhibit a selective defect in protein degradation it is thought that uba-ubl proteins exhibit some degree of selectivity in targeting specific substrates to the proteasome. we hypothesized that the decrease of ddi binding to proteasomes in an rpn1-d517a mutant might therefore result in turnover defects of substrates that are particularly reliant on ddi in agreement with the normal binding of rad and dsk to the proteasome in an rpn1-d517a single mutant, no defect was seen in turnover of the rad23/dsk2-dependent substrate, cpy*, or the dsk substrate kre. however, when we tested the ddi1-dependent substrate, ufo, we saw nearly complete stabilization in comparison to a wild type strain. note that the initial levels of plasmid encoded gst-ufo were higher in rpn1-d517a compared to ddi1Δ cells. this was seen in two of three replicates. we do not know the basis for this. in agreement with the turnover data, we observed that over-expression of gst-ufo was toxic to ddi1Δ and rpn1-d517a cells but not wild type cells. this effect was exquisitely specific--neither rpn13Δ nor any other mutation in rpn that we tested conferred sensitivity to over-expression of dst-ufo discussion of the three uba-ubl shuttle receptors linked to the proteasome, ddi is the least studied and perhaps the most controversial. prior data have established that ddi binds polyubiquitin, albeit with lower affinity than rad and dsk. however, while some studies report a physical interaction of ddi with rpn or the intact proteasome, there are a few reports that question the capacity for ddi to bind the proteasome or rpn. the disparity in these reports may be due to the qualitative nature of immunoprecipitation experiments and the rapid dynamics of ubl binding to and dissociation from the proteasome. ddi has the most divergent ubl domain among the known uba-ubl proteins, and hence, may have the weakest affinity interaction with the proteasome. we have shown that ddi is recovered with proteasomes immunoprecipitated from yeast cells, binds rpn in a yeast two-hybrid assay, and binds to the proteasome in an in vitro pull-down assay. we have further validated these results by identifying an rpn mutation that is selectively defective in binding ddi and stabilizes the ddi1-dependent proteasome substrate ufo hence, we conclude that ddi does indeed interact with the proteasome in a specific and functionally-relevant manner. if ddi binds the proteasome more weakly than other uba-ubl proteins, which seems likely, it could explain why the d517a and k484a mutations reported here selectively disrupt interaction of ddi with the proteasome. our study highlights the layered complexity of the interaction of shuttle proteins with the proteasome. with a single alanine substitution in the highly conserved d residue of rpn we were able to significantly reduce the binding of ddi to the proteasome. however, the interaction of other uba-ubl proteins with the proteasome appears to be more complex. recovery of dsk with proteasomes was only mildly diminished in an rpn1-d517a mutant that also lacked rpn or the ubiquitin interaction motifs of both rpn and rpn meanwhile, recovery of rad was not affected appreciably by any mutation in rpn analyzed during the course of this work. there are two possible explanations of these results. on the one hand, it is possible that the domains of these proteins have a gradient of affinity for rpn with ddi being the weakest binder and rad the strongest. in this scenario, rpn1-d517a may be a hypomorph that only modestly perturbs the ubl docking site, such that only the weakest binder is excluded. we attempted to test this hypothesis by making numerous combinatorial mutations,, none of which exhibited a substantially greater ubl binding defect than the d517a or k484a alleles. thus, we do not favor the hypothesis that it is possible to disrupt recruitment of rad and dsk by mutating a single binding patch on rpn on the other hand, our model for ubl docking to the proteasome suggests that it is possible that ddi uses only a single mechanism to bind the proteasome, whereas, in line with published reports, rad and dsk may use multiple mechanisms and thus are more resistant to mutation. our observation that reduction of dsk binding was only seen in an rpn1-d517a rpn13Δ double mutant and more strikingly in a rpn1-d517a rpn13Δ rpn10-uim supports the idea that dsk may be tethered to the proteasome by either rpn rpn or rpn. the failure to see a significant reduction in binding of rad in any single or double mutant may be due to there being multiple independent docking sites for rad on the proteasome, although it should be noted that all of these docking sites appear to rely on the ubl domain. other studies have shown that ubp may bind proteasome lid proteins while rad may also bind rpt, and that even ub chains bound to rad may contribute to its proteasome binding. biochemical data suggest that human rad is recruited to the proteasome by the uim domain of rpn, and that even yeast rad can bind rpn but it should be noted that this hypothesis has not been tested by genetic manipulation of rpn in cells. clearly, more work is needed to unravel the mechanisms underlying recruitment of the ubl domain proteins of rad dsk and ubp to the proteasome.  the current study identifies residues in the lrr domain of rpn that contribute to shuttle receptor docking. we validate ddi as a proteasomal shuttle receptor whose stable binding to the proteasome depends on rpn residue d consistent with this, d is also important for the degradation of a ddi1-dependent substrate. we also show that in the absence of rpn or the dual absence of the ubiquitin binding domains of rpn and rpn mutation of the d or k residues reduces the association of dsk with the proteasome. 
here we demonstrate that delivery of ub conjugates and docking of ddi to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved d residue of rpn moreover, degradation of the ddi1-dependent proteasome substrate, ufo is blocked in rpn1-d517a yeast cells. to shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in rpn that disrupt its binding to uba-ubl proteins. our studies suggest that different uba-ubl proteins are recruited to the proteasome by distinct mechanisms.
 the proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin chain appendage. these ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors rpn rpn or rpt or are shuttled to the proteasome by rad dsk or ddi the latter proteins share an ub association domain for binding poly-ub chains and an ub-like-domain for binding to the proteasome. it has been proposed that shuttling receptors dock on the proteasome via rpn but the precise nature of the docking site remains poorly defined.  to shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in rpn that disrupt its binding to uba-ubl proteins. here we demonstrate that delivery of ub conjugates and docking of ddi to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved d residue of rpn moreover, degradation of the ddi1-dependent proteasome substrate, ufo is blocked in rpn1-d517a yeast cells. by contrast, rad recruitment to the proteasome is not affected by rpn1-d517a.  these studies provide insight into the mechanism by which the uba-ubl protein ddi is recruited to the proteasome to enable ub-dependent degradation of its ligands. our studies suggest that different uba-ubl proteins are recruited to the proteasome by distinct mechanisms. 