BACKGROUND
in eukaryotes, the 26s proteasome handles the majority of regulated proteolysis and is pivotal for the proper function of the cell  <cit> .
most proteins that are targeted to the proteasome for degradation are first modified by the ubiquitin  system  <cit> .
specifically, successive ub molecules join to form a ub chain on the substrates.
how the ubiquitylated substrate is then delivered to and degraded by the 26s proteasome remains elusive  <cit> .
it is now widely accepted that ub-binding proteins  play important roles in facilitating substrate transfer to the proteasome.
these proteins have distinct substrate specificity and functionally cooperate for substrate proteolysis  <cit> .
however, how any one of these proteins works in vivo remains largely unknown  <cit> .
rad <dig> belongs to a family of proteins that contain both the ub-associated  domain and a ub-like  motif  <cit> .
the uba motif binds specifically to ub chain/conjugates in vivo and in vitro  <cit> .
the ubl motif directly binds the proteasome  <cit> .
through these domain-mediated interactions, uba/ubl proteins link ubiquitylated proteins to the proteasome.
in the budding yeast saccharomyces cerevisiae, there are three uba/ubl-containing proteins, rad <dig>  dsk <dig> and ddi <dig>  which have been implicated in ub-mediated proteolysis.
the loss of s. cerevisiae rad <dig> and dsk <dig> leads to the stabilization of an artificially designed ub-fusion substrate ubv76-v-βgal  <cit> , the cell cycle inhibitor far <dig>  <cit> , misfolded protein cpy*  <cit> , an er protein hmg <dig>  <cit> , and the homologues of rad <dig> are involved in the degradation of the cdk inhibitor rum <dig>  <cit> , pax <dig>  <cit>  and the tumor suppressor p <dig>  <cit> .
consistent with the role of rad <dig> and dsk <dig> in post-ubiquitylation events, the stabilized substrates in rad23Δ dsk2Δ mutant cells are fully ubiquitylated.
rad <dig> and dsk <dig> play overlapping functions since cells lacking both rad <dig> and dsk <dig> are more sensitive to various stress conditions  than either single mutant  <cit> .
however, the biological pathways that require the functioning of rad <dig> and dsk <dig> remain to be identified.
not all proteolytic substrates are degraded by the rad23/dsk2-dependent pathway.
other ub-binding proteins, rpn <dig>  rpn <dig>  and the cdc48-ufd1-npl <dig> complex also promote proteasome-mediated proteolysis and exhibit distinct substrate specificity  <cit> .
for example, the cell cycle protein clb <dig> requires rpn <dig>  but not rad <dig> for its degradation.
and deg1-gfp is degraded by the cdc48/ufd <dig> pathway, independent of rpn <dig> and rad <dig>  the principle underlying the division of labor among these ub-binding proteins is far from clear.
a key to unraveling the functions of these ub receptors  is to identify their cellular substrates and biological functions.
for example, using mass spectrometry, more than  <dig> proteins were shown to be preferentially accumulated in cells lacking rpn <dig>  <cit> .
these putative rpn <dig> substrates are involved in cell cycle control, protein transport, metabolism, and transcription.
here, we employ a new method to identify the biological function of rad <dig> and dsk <dig>  the lack of overt phenotypes in rad23Δ dsk2Δ mutant cells likely reflects that redundant pathways are employed in buffering against mutations.
using a synthetic enhancement approach, we have isolated  <dig> genes that when overexpressed cause slow growth in rad23Δ dsk2Δ mutants.
our data expand the physiological processes that involve rad <dig> and dsk <dig>  furthermore, we show that two of the encoded proteins isolated by this method are ubiquitylated and degraded by the dsk2-dependent pathway.
our results also suggest that the synthetic dosage lethality screen can be used to isolate physiological targets of a specific proteolytic pathway.
methods
strains, media and plasmids
yeast cultures were grown in rich  or synthetic media containing standard ingredients and 2% glucose , 2% raffinose , or 2% raffinose + 2% galactose .
the synthetic genetic array  compatible s288c strain y <dig>  and isogenic rad23Δ dsk2Δ  cells were used for all assays involving the deletions of rad <dig> and dsk <dig>  strains jd <dig> , jd <dig>  and ufd1- <dig> were obtained from dr. alex varshavsky   <cit> .
haploid strains bearing rad23Δ, dsk2Δ, ddi1Δ, rpn10Δ, rpn13Δ, kre22Δ, in the by <dig> background were obtained from open biosystems .
for the synthetic dosage lethality  screens, haploids were selected on synthetic dextrose medium , supplemented with the following antibiotics  <dig> mg/l clonnat ,  <dig> mg/l geneticin ,  <dig> mg/l l-canavanine  and  <dig> mg/l s--l-cysteine hydrochloride .
the plasmids expressing the proteasomal substrates arg-βgal, scc <dig>  or ubv76-v-βgal, ricin a chain  have been previously described  <cit>  and substrate expression are regulated by the gal <dig> promoter.
the pegh plasmids, which contain an individual yeast gene tagged with both glutathione s-transferase  protein and his <dig> at its amino-terminus, were previously described  <cit> .
the movable orf  clones were purchased from open biosystems .
the plasmid expressing ha-ub was obtained from dr. mary ann osley .
genome-wide synthetic dosage lethal screen
the ura3-marked overexpression library and the screening procedures used in this study were previously described  <cit> .
briefly, two isogenic synthetic genetic array -compatible strains, y <dig> and rad23Δ dsk2Δ, were mated to an ordered yeast array expressing  <dig> unique galactose-inducible genes.
the arrayed strains were subjected to diploid selection, sporulation and two rounds of haploid selection  <cit> , to give rise to an output array of duplicated colonies carrying the desirable natr-marked deletion and one unique galactose-inducible gene.
the haploid arrays were finally replica-pinned onto media containing 2% glucose  or 2% galactose .
the colonies on the glucose and galactose plates were photographed after two and three days of incubation, respectively.
overexpressed genes that uniquely caused a reduction in colony size of more than 20% in the rad23Δ dsk2Δ background were considered for downstream analysis.
expression shut-off assay and proteasome inhibition treatment
yeast cells carrying plasmids expressing hexahistidine  and gst double-tagged proteins from the gal <dig> promoter were grown at 30°c to an od <dig> of approximately  <dig> in sr-ura medium with auxotrophic supplements and 2% raffinose as the carbon source.
protein expression was induced with 2% galactose for  <dig> h and then repressed by the addition of 2% glucose.
cycloheximide  was also added to stop translation.
samples were withdrawn at the indicated time points and harvested by centrifugation.
proteins were extracted by glass bead lysis of cells, processed for immunoprecipitation with glutathione sepharose beads , and resolved by 10% sds-page.
immunoblots were probed with anti-his <dig> antibody  followed by detection with goat anti-mouse horseradish peroxidase  conjugate using chemiluminescent reagents .
the stable protein rpt <dig> was used as a loading control in the expression shutoff experiments.
proteasome inhibition was performed as described  <cit> .
briefly, yeast cells were grown in synthetic media using proline as the only nitrogen source.
sds  was added to the media  <dig> h before galactose induction.
mg <dig>  was added  <dig> min before the addition of glucose.
samples were collected at indicated time points and processed as described above.
detection of ubiquitylated substrates
yeast cells expressing gst-tagged substrates and ha-tagged ub or myc-tagged ub were grown in galactose-containing sg medium to an od <dig> of approximately  <dig>  cells were lysed with glass beads and immunoprecipitated with glutathione sepharose beads for  <dig> h at 4°c.
the immunoprecipitates were resolved by sds-page, transferred to polyvinylidene fluoride  membrane, and immunoblotted with anti-ha antibody , followed by anti-mouse hrp conjugates and ecl reagents.
RESULTS
accumulation of proteasomal substrates in degradation mutants can lead to growth retardation
we set out to look for physiological substrates of rad <dig> and dsk <dig>  many strategies have been developed in the past to isolate the substrates of a particular ub/proteasome pathway with varying degrees of success.
most of these methods explore the facts that substrates directly bind to the degradation machinery and/or substrates are ubiquitylated.
two challenges often encountered in these endeavors are the transient nature of the interaction between substrates and degradation components, and low levels of substrate expression in vivo.
to identify the physiological functions of rad <dig> and dsk <dig>  we employed a synthetic dosage lethality screen  <cit> .
the premise of sdl is that increased levels of a protein have little toxic effect on the growth of a wild-type strain but may cause growth retardation or even lethality in a mutant strain.
previous studies suggest that this strategy can be adapted to isolate substrates of the ub/proteasome system.
for example, overexpression of several cell cycle proteins  in proteolysis-deficient cells  led to extremely slow cell growth .
the growth defect induced by substrate scc <dig> overexpression is particularly striking in the case of ubr1Δ cells lacking the e <dig> enzyme of the n-end rule degradation pathway, since the absence of ubr <dig> alone does not significantly alter cell growth  <cit> .
we then wondered whether the toxicity might be related to the physiological functions of the substrates.
in other words, would accumulation of large amounts of any substrate lead to growth retardation in proteolysis-deficient cells?
we used an artificially designed n-end rule substrate arg-βgal, which contains βgalactosidase from e. coli fused to a destabilizing residue arg  <cit> .
deletion of ubr <dig> leads to the accumulation of arg-βgal, which does not affect cell growth .
similarly, we found that compromised degradation of another model substrate of the proteasome ubv76-val-βgal does not elicit toxicity in rad23Δ dsk2Δ mutants   <cit> , indicating that the toxicity triggered by substrate accumulation may depend on the physiological function of the target protein.
these results further suggest that the sdl screen can identify a subset of substrates of a given proteolytic pathway.
identification of yeast genes which cause slower growth in rad <dig> dsk <dig> mutant cells upon overexpression
the genome-wide sdl screen was carried out as previously described  <cit> .
more than  <dig> yeast genes fused to both glutathione s-transferase and his <dig> tags were separately introduced into wild-type or rad23Δ dsk2Δ mutant cells  <cit> .
we compared the growth of wild type vs. rad23Δ dsk2Δ cells that expressed each of these yeast genes from a strong, regulatable gal <dig> promoter.
the expression of these genes was repressed in glucose containing media, but induced in galactose-containing media.
haploids carrying these plasmids were pinned onto both sd  and sg  plates.
following incubation for two to three days at 30°c , the colony sizes of these plates were scored using a program developed in dr. boone's lab .
upon their overexpression, a total of  <dig> genes were found to preferentially cause slower growth in rad23Δ dsk2Δ mutant cells.
sic <dig> and far <dig>  two known substrates of rad <dig> and dsk <dig>  <cit> , were not isolated as positives in our screen because their overexpression leads to severe toxicity even in wild-type cells , suggesting that genes, which severely reduce growth rate in wild-type cells may not be recovered in this type of sdl screen.
as is common in nearly all screens, we expected our approach to yield false positives, especially since a low cut-off  is used to define a hit.
the 20% cut-off ensures that most of the toxic genes are efficiently captured in our screens .
to eliminate false hits, we performed a serial spotting assay with each candidate identified.
more specifically, wild-type or rad23Δ dsk2Δ mutant cells containing the candidate open reading frame  regulated by the gal <dig> promoter were grown in non-inducible raffinose media to a similar od <dig>  and then spotted onto sd and sg plates in five-fold serial dilutions.
the growth rates of wild-type and rad23Δ dsk2Δ mutant cells bearing a vector plasmid are similar under the condition .
of the  <dig> genes identified in the primary screen, eleven genes were confirmed to cause noticeably slower growth in mutant cells than in wild-type cells upon overexpression .
whereas two identified genes  have not been functionally characterized, the other nine genes are involved in a variety of cellular processes .
aad <dig> encodes an aryl alcohol dehydrogenase important for amino acid catabolism.
mss <dig> and rfx <dig> are transcription factors involved in the regulation of invasive growth and in dna damage responses, respectively.
ppg <dig> has protein phosphatase activity and is involved in glycogen accumulation.
systematic deletion studies suggest that kre <dig>  is involved in glycogen accumulation  <cit> , mitochondrial distribution and morphology  <cit> , and is important in resistance to dna damaging agents   <cit> .
vph <dig> is a subunit of the vacuolar h-atpase essential for vacuolar acidification.
ste <dig> functions as the β subunit of the g protein that mediates pheromone-induced signal transduction.
dtr <dig> encodes the dityrosine transporter involved in the maturation of the spore wall.
it is worth noting that overexpression of spc <dig>  a component of the spindle pole body  <cit> , causes more severe growth retardation in rad23Δ dsk2Δ mutant cells than in wild-type cells , consistent with previous findings that suggest a role of rad <dig> and dsk <dig> in spindle function  <cit> .
the degradation of kre <dig> and yll030c requires rad23/dsk2
as reasoned above, accumulation of a large amount of substrates can lead to toxicity in cells defective in proteolysis .
we examined the stability of the proteins encoded by the eleven isolated genes in wild-type or mutant cells by an expression shut-off assay.
two proteins, kre <dig> and yll030c are rapidly degraded in wild-type cells, but significantly stabilized in cells lacking rad <dig> and dsk <dig> , suggesting that they are degraded by the rad23/dsk <dig> pathway.
deletion of rad <dig> and dsk <dig> does not significantly alter the half-life of the other nine proteins identified by our screen .
previously, we demonstrated that rad <dig> works with two different co-factors ufd <dig>  and png <dig>  to degrade distinct substrates  <cit> .
here, we examined whether kre <dig> or yll030c turn-over requires ufd <dig> or png <dig>  deletion of ufd <dig> does not alter the degradation of kre <dig> or yll030c .
the png1-rad <dig> complex is involved in degrading glycosylated proteins  <cit> .
we found that neither protein is glycosylated , suggesting that kre <dig> and yll030c are not png <dig> substrates.
it is likely that these substrates would require co-factors other than ufd <dig> and png <dig> for their degradation.
since these substrates are tagged with gst moiety at their n-terminus, one concern of our results is that the degradation of these proteins may be altered by the gst fusion.
to this end, we obtained the morf plasmids expressing yll030c and kre <dig>  which are tagged at their carboxyl-terminus with both his <dig> and the igg-binding site from protein a  that theoretically could also affect protein degradation.
as shown in figure  <dig>  yll030c and kre <dig> are still degraded in rad23/dsk2-dependent manner , suggesting that the involvements of rad <dig> and dsk <dig> in kre <dig> and yll030c degradation are not due to artifacts caused by gst tags.
overexpression of these morf plasmids also led to growth retardation in rad23Δ dsk2Δ mutant cells .
kre <dig> and yll030c are ubiquitylated, proteasomal substrates
are these newly identified rad23/dsk <dig> substrates destroyed by the proteasome or some other proteolytic activity ?
to assess the involvement of the proteasome in the degradation of kre <dig> or yll030c, we carried out a simple proteasome inhibition assay  <cit> .
we found that the degradation of kre <dig> or yll030c is impaired upon mg <dig> treatment , suggesting that these proteins are degraded by the proteasome.
most, but not all, proteasomal substrates are ubiquitylated.
the function of rad23/dsk <dig> in proteolysis requires their uba domains, which bind to ub  <cit> .
to determine whether the newly identified substrates are ubiquitylated, we co-transformed the plasmid expressing the gst- and his6-tagged substrate with the plasmid bearing ha-tagged ub into wild-type or rad23Δ dsk2Δ mutant cells.
gst-tagged substrates were immunoprecipitated from cellular lysates with gst beads, and processed for western blotting with anti-ha antibody to detect ubiquitylated substrate.
we found that both kre <dig> and yll030c are ubiquitylated .
consistent with the function of rad <dig> and dsk <dig> at a pre-proteasome but post-ubiquitylation step  <cit> , much more ubiquitylated species were detected in the rad23Δ dsk2Δ mutants than in wild type cells .
the involvement of specific ub receptor for the degradation of kre <dig> or yll030c
ub-binding proteins that function in proteasome-mediated proteolysis include three uba/ubl proteins , two proteasome-associated factors rpn <dig> and rpn <dig>  and the ufd1-cdc48-npl <dig> complex  <cit> .
these ub-receptors have different substrates and sometimes carry out overlapping functions  <cit> .
we examined the stability of gst-tagged kre <dig> and yll030c in yeast cells lacking individual ub receptor .
both dsk <dig> and ufd <dig> are required for efficient degradation of yll030c .
interestingly, dsk <dig> is the only ub receptor involved in kre <dig> degradation .
furthermore, consistent with the restriction of dsk <dig> by rpn <dig>  <cit> , kre <dig> and yll030c are degraded faster in rpn10Δ cells than in wild-type cells .
kre <dig> overexpression suppresses dsk2-induced toxicity
to our knowledge, kre <dig> is the first substrate that requires only dsk <dig> but none of the other ub receptors for its degradation .
supporting the direct link between dsk <dig> and kre <dig>  we detected the dsk2-kre <dig> interaction by co-immunoprecipitations .
it will be important to determine the specific domains of dsk <dig> involved in kre22-binding and degradation, and the role of ubiquitylation in the dsk2-kre <dig> interaction.
since little is known about dsk2-specific substrates, we examined the ub linkage assembled onto kre <dig>  lys48- and lys63-linked chains were recently linked to the dsk <dig> pathway  <cit> .
specifically, we employed ub mutants  that had either lys <dig> or lys <dig> replaced with arg, which in turn blocked ub conjugation to these lysine residues  <cit> .
we found that kre <dig> ubiquitylation was abolished by lys48arg mutation , suggesting that the ub chain attached to kre <dig> contained lys <dig> linkage.
among ub-binding proteins involved in proteasome-mediated proteolysis, dsk <dig> has the unique property that its overexpression caused toxicity  <cit> .
we examined whether the deletion or overexpression of kre <dig> could counteract overdose of dsk <dig> .
interestingly, increased expression of kre <dig> partially suppressed dsk2-induced toxicity .
we then asked whether other proteasomal substrates could keep dsk <dig> in check.
we employed rta, a rad <dig>  but not dsk <dig> substrate, and ubv76-val-βgal that is regulated by both rad <dig> and dsk <dig>  interestingly, the suppression of dsk <dig> is substrate specific since the overexpression of rta or ubv76-val-βgal could not overcome dsk2-triggered toxicity .
yll030c overexpression also partially suppressed dsk2-induced growth retardation
discussion
we describe herein the use of a large-scale sdl screen as a new approach to identify proteolytic targets of the rad23/dsk2-dependent pathway.
although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.
for example, the first genetically defined ub-dependent proteolytic pathway  was discovered in the mid-1980s  <cit> .
despite over a decade of intense efforts, the first cellular substrate of the n-end rule pathway was identified fortuitously.
a key cell cycle protein, scc <dig> is cleaved at the metaphase-anaphase transition  <cit>  and the resulting carboxyl-terminal fragment is destroyed by the n-end rule pathway to maintain chromosome stability  <cit> .
interestingly, overexpression of scc <dig> leads to lethality in mutant cells lacking the n-end rule e <dig> enzyme .
this finding highlights the difficulty in obtaining proteolytic substrates and also suggests that an sdl screen could be effective for substrate identification.
an sdl approach was first employed to uncover genetic interactions in dna replication or chromosome segregation  <cit> .
more recently, it was demonstrated that sdl screens could be used to identify novel targets of a protein kinase   <cit> .
our study further extends the effectiveness of the sdl screen to the ub/proteasome system.
the advantages of sdl as a screening tool for proteolytic substrate identification include that the assay is performed in vivo, and the protein expression levels are enhanced by the strong promoter .
like other approaches for target discovery, sdl screens have limitations.
of approximately  <dig> yeast genes we screened, only two dsk <dig> substrates were isolated.
several factors may contribute to the high false negative level: 1) redundancy of various substrate delivery pathways  <cit> .
several known dsk2/rad <dig> substrates   <cit>  were not recovered in this screen.
in the absence of dsk2/rad <dig>  other ub adaptor molecules  may maintain sufficient proteolysis  <cit> ; 2) genes that lead to severe growth defects in wild-type cells with increased dosage are likely to be excluded from such analysis; 3) not all substrates would elicit enhanced fitness defects in proteolysis mutants ; 4) the screen is an indirect way for target discovery.
large number of false positives, partly due to a low colony size cutoff, may mask some true positives; 5) screening strategy and criteria for target identification may need further refinement.
for example, the rapid turnover or absence of particular substrates might be a requirement under very specific environmental or physiological conditions, which were not covered by our standardized screening protocol .
nevertheless, given the difficulty involved in substrate isolation, we believe that sdl provides another way to delineate the functional relationship between a substrate and the specific degradation pathway.
to our knowledge, gst-kre <dig> is the first substrate that requires only dsk <dig>  but not other ub receptors  for degradation .
interestingly, overexpressed kre <dig> but not other proteasomal substrates suppresses dsk2-induced toxicity .
although the precise mechanism underlying the dsk2-triggered toxic effect is not clear  <cit> , one possible explanation is that excessive dsk <dig> may engage in the abnormal activities that interfere with cell growth.
and increased amount of specific substrates may keep dsk <dig> in check.
our results suggest that kre <dig> could serve as an important tool to understand the dsk2-dependent proteolysis.
most proteins identified are not direct proteolytic substrates of rad <dig> and dsk <dig>  this is not unexpected since other genes that function in the linear pathway of rad23/dsk <dig> target may also induce toxicity in rad <dig> dsk <dig> mutant cells upon their overexpression.
for example, in a separate study on the function of rad <dig> and dsk <dig>  we identified a cell cycle protein involved in the spindle pole body  duplication as a degradation target of rad <dig> and dsk <dig> .
interestingly, this protein is known to act upstream of spc <dig>  a component of the spb  <cit> .
it is likely that increased levels of spc <dig> mimic the accumulation of this rad23/dsk <dig> target involved in spindle regulation to affect spb duplication, which in turn triggers growth retardation in rad <dig> dsk <dig> mutant cells.
therefore, although the other eight proteins identified by our screen are not regulated directly by rad <dig> and dsk <dig>  we suspect that these proteins work with rad23/dsk <dig> targets in vivo and cellular responses to their overexpression require the function of rad <dig> and dsk <dig> .
CONCLUSIONS
in conclusion, as proof-of-concept, we show that an sdl screen offers an effective, complementary method to elucidating biological functions of proteolytic pathways.
dsk <dig> was first identified  <dig> years ago in a genetic screen for genes involved in spb duplication  <cit> .
to our knowledge, the spc98-induced toxicity in rad <dig> dsk <dig> mutant cells is the second piece of evidence linking dsk <dig> and rad <dig> to spb duplication, which will help us to unravel the specific role of rad <dig> and dsk <dig> in this process.
it is also conceivable that the sdl screen can be adapted to identify relevant ubiquitylation enzymes involved in substrate turnover.
the creation of rnai knock-down libraries and orf overexpression collections would extend the use of sdl screens in the ub/proteasome system to higher eukaryotes.
abbreviations
spb: spindle pole body; ub: ubiquitin; uba: ubiquitin-associated; ubl: ubiquitin-like; sdl: synthetic dosage lethality; his6: hexahistidine; gst: glutathione stransferase.
authors' contributions
cl, dvd and yl performed the experiments.
ba and hr conceived the study and designed the experiments.
cl, dvd, ba and hr wrote the manuscript.

 in eukaryotes, the 26s proteasome handles the majority of regulated proteolysis and is pivotal for the proper function of the cell. most proteins that are targeted to the proteasome for degradation are first modified by the ubiquitin system. specifically, successive ub molecules join to form a ub chain on the substrates. how the ubiquitylated substrate is then delivered to and degraded by the 26s proteasome remains elusive. it is now widely accepted that ub-binding proteins play important roles in facilitating substrate transfer to the proteasome. these proteins have distinct substrate specificity and functionally cooperate for substrate proteolysis. however, how any one of these proteins works in vivo remains largely unknown. rad belongs to a family of proteins that contain both the ub-associated domain and a ub-like motif. the uba motif binds specifically to ub chain/conjugates in vivo and in vitro. the ubl motif directly binds the proteasome. through these domain-mediated interactions, uba/ubl proteins link ubiquitylated proteins to the proteasome. in the budding yeast saccharomyces cerevisiae, there are three uba/ubl-containing proteins, rad dsk and ddi which have been implicated in ub-mediated proteolysis. the loss of s. cerevisiae rad and dsk leads to the stabilization of an artificially designed ub-fusion substrate ubv76-v-βgal, the cell cycle inhibitor far, misfolded protein cpy*, an er protein hmg, and the homologues of rad are involved in the degradation of the cdk inhibitor rum, pax and the tumor suppressor p. consistent with the role of rad and dsk in post-ubiquitylation events, the stabilized substrates in rad23Δ dsk2Δ mutant cells are fully ubiquitylated. rad and dsk play overlapping functions since cells lacking both rad and dsk are more sensitive to various stress conditions than either single mutant. however, the biological pathways that require the functioning of rad and dsk remain to be identified. not all proteolytic substrates are degraded by the rad23/dsk2-dependent pathway. other ub-binding proteins, rpn rpn and the cdc48-ufd1-npl complex also promote proteasome-mediated proteolysis and exhibit distinct substrate specificity. for example, the cell cycle protein clb requires rpn but not rad for its degradation. and deg1-gfp is degraded by the cdc48/ufd pathway, independent of rpn and rad the principle underlying the division of labor among these ub-binding proteins is far from clear. a key to unraveling the functions of these ub receptors is to identify their cellular substrates and biological functions. for example, using mass spectrometry, more than proteins were shown to be preferentially accumulated in cells lacking rpn. these putative rpn substrates are involved in cell cycle control, protein transport, metabolism, and transcription. here, we employ a new method to identify the biological function of rad and dsk the lack of overt phenotypes in rad23Δ dsk2Δ mutant cells likely reflects that redundant pathways are employed in buffering against mutations. using a synthetic enhancement approach, we have isolated genes that when overexpressed cause slow growth in rad23Δ dsk2Δ mutants. our data expand the physiological processes that involve rad and dsk furthermore, we show that two of the encoded proteins isolated by this method are ubiquitylated and degraded by the dsk2-dependent pathway. our results also suggest that the synthetic dosage lethality screen can be used to isolate physiological targets of a specific proteolytic pathway. methods strains, media and plasmids yeast cultures were grown in rich or synthetic media containing standard ingredients and 2% glucose, 2% raffinose, or 2% raffinose + 2% galactose. the synthetic genetic array compatible s288c strain y and isogenic rad23Δ dsk2Δ cells were used for all assays involving the deletions of rad and dsk strains jd, jd and ufd1- were obtained from dr. alex varshavsky. haploid strains bearing rad23Δ, dsk2Δ, ddi1Δ, rpn10Δ, rpn13Δ, kre22Δ, in the by background were obtained from open biosystems. for the synthetic dosage lethality screens, haploids were selected on synthetic dextrose medium, supplemented with the following antibiotics mg/l clonnat, mg/l geneticin, mg/l l-canavanine and mg/l s--l-cysteine hydrochloride. the plasmids expressing the proteasomal substrates arg-βgal, scc or ubv76-v-βgal, ricin a chain have been previously described and substrate expression are regulated by the gal promoter. the pegh plasmids, which contain an individual yeast gene tagged with both glutathione s-transferase protein and his at its amino-terminus, were previously described. the movable orf clones were purchased from open biosystems. the plasmid expressing ha-ub was obtained from dr. mary ann osley. genome-wide synthetic dosage lethal screen the ura3-marked overexpression library and the screening procedures used in this study were previously described. briefly, two isogenic synthetic genetic array -compatible strains, y and rad23Δ dsk2Δ, were mated to an ordered yeast array expressing unique galactose-inducible genes. the arrayed strains were subjected to diploid selection, sporulation and two rounds of haploid selection, to give rise to an output array of duplicated colonies carrying the desirable natr-marked deletion and one unique galactose-inducible gene. the haploid arrays were finally replica-pinned onto media containing 2% glucose or 2% galactose. the colonies on the glucose and galactose plates were photographed after two and three days of incubation, respectively. overexpressed genes that uniquely caused a reduction in colony size of more than 20% in the rad23Δ dsk2Δ background were considered for downstream analysis. expression shut-off assay and proteasome inhibition treatment yeast cells carrying plasmids expressing hexahistidine and gst double-tagged proteins from the gal promoter were grown at 30°c to an od of approximately in sr-ura medium with auxotrophic supplements and 2% raffinose as the carbon source. protein expression was induced with 2% galactose for h and then repressed by the addition of 2% glucose. cycloheximide was also added to stop translation. samples were withdrawn at the indicated time points and harvested by centrifugation. proteins were extracted by glass bead lysis of cells, processed for immunoprecipitation with glutathione sepharose beads, and resolved by 10% sds-page. immunoblots were probed with anti-his antibody followed by detection with goat anti-mouse horseradish peroxidase conjugate using chemiluminescent reagents. the stable protein rpt was used as a loading control in the expression shutoff experiments. proteasome inhibition was performed as described. briefly, yeast cells were grown in synthetic media using proline as the only nitrogen source. sds was added to the media h before galactose induction. mg was added min before the addition of glucose. samples were collected at indicated time points and processed as described above. detection of ubiquitylated substrates yeast cells expressing gst-tagged substrates and ha-tagged ub or myc-tagged ub were grown in galactose-containing sg medium to an od of approximately cells were lysed with glass beads and immunoprecipitated with glutathione sepharose beads for h at 4°c. the immunoprecipitates were resolved by sds-page, transferred to polyvinylidene fluoride membrane, and immunoblotted with anti-ha antibody, followed by anti-mouse hrp conjugates and ecl reagents.  accumulation of proteasomal substrates in degradation mutants can lead to growth retardation we set out to look for physiological substrates of rad and dsk many strategies have been developed in the past to isolate the substrates of a particular ub/proteasome pathway with varying degrees of success. most of these methods explore the facts that substrates directly bind to the degradation machinery and/or substrates are ubiquitylated. two challenges often encountered in these endeavors are the transient nature of the interaction between substrates and degradation components, and low levels of substrate expression in vivo. to identify the physiological functions of rad and dsk we employed a synthetic dosage lethality screen. the premise of sdl is that increased levels of a protein have little toxic effect on the growth of a wild-type strain but may cause growth retardation or even lethality in a mutant strain. previous studies suggest that this strategy can be adapted to isolate substrates of the ub/proteasome system. for example, overexpression of several cell cycle proteins in proteolysis-deficient cells led to extremely slow cell growth. the growth defect induced by substrate scc overexpression is particularly striking in the case of ubr1Δ cells lacking the e enzyme of the n-end rule degradation pathway, since the absence of ubr alone does not significantly alter cell growth. we then wondered whether the toxicity might be related to the physiological functions of the substrates. in other words, would accumulation of large amounts of any substrate lead to growth retardation in proteolysis-deficient cells? we used an artificially designed n-end rule substrate arg-βgal, which contains βgalactosidase from e. coli fused to a destabilizing residue arg. deletion of ubr leads to the accumulation of arg-βgal, which does not affect cell growth. similarly, we found that compromised degradation of another model substrate of the proteasome ubv76-val-βgal does not elicit toxicity in rad23Δ dsk2Δ mutants, indicating that the toxicity triggered by substrate accumulation may depend on the physiological function of the target protein. these results further suggest that the sdl screen can identify a subset of substrates of a given proteolytic pathway. identification of yeast genes which cause slower growth in rad dsk mutant cells upon overexpression the genome-wide sdl screen was carried out as previously described. more than yeast genes fused to both glutathione s-transferase and his tags were separately introduced into wild-type or rad23Δ dsk2Δ mutant cells. we compared the growth of wild type vs. rad23Δ dsk2Δ cells that expressed each of these yeast genes from a strong, regulatable gal promoter. the expression of these genes was repressed in glucose containing media, but induced in galactose-containing media. haploids carrying these plasmids were pinned onto both sd and sg plates. following incubation for two to three days at 30°c, the colony sizes of these plates were scored using a program developed in dr. boone's lab. upon their overexpression, a total of genes were found to preferentially cause slower growth in rad23Δ dsk2Δ mutant cells. sic and far two known substrates of rad and dsk, were not isolated as positives in our screen because their overexpression leads to severe toxicity even in wild-type cells, suggesting that genes, which severely reduce growth rate in wild-type cells may not be recovered in this type of sdl screen. as is common in nearly all screens, we expected our approach to yield false positives, especially since a low cut-off is used to define a hit. the 20% cut-off ensures that most of the toxic genes are efficiently captured in our screens. to eliminate false hits, we performed a serial spotting assay with each candidate identified. more specifically, wild-type or rad23Δ dsk2Δ mutant cells containing the candidate open reading frame regulated by the gal promoter were grown in non-inducible raffinose media to a similar od and then spotted onto sd and sg plates in five-fold serial dilutions. the growth rates of wild-type and rad23Δ dsk2Δ mutant cells bearing a vector plasmid are similar under the condition. of the genes identified in the primary screen, eleven genes were confirmed to cause noticeably slower growth in mutant cells than in wild-type cells upon overexpression. whereas two identified genes have not been functionally characterized, the other nine genes are involved in a variety of cellular processes. aad encodes an aryl alcohol dehydrogenase important for amino acid catabolism. mss and rfx are transcription factors involved in the regulation of invasive growth and in dna damage responses, respectively. ppg has protein phosphatase activity and is involved in glycogen accumulation. systematic deletion studies suggest that kre is involved in glycogen accumulation, mitochondrial distribution and morphology, and is important in resistance to dna damaging agents. vph is a subunit of the vacuolar h-atpase essential for vacuolar acidification. ste functions as the β subunit of the g protein that mediates pheromone-induced signal transduction. dtr encodes the dityrosine transporter involved in the maturation of the spore wall. it is worth noting that overexpression of spc a component of the spindle pole body, causes more severe growth retardation in rad23Δ dsk2Δ mutant cells than in wild-type cells, consistent with previous findings that suggest a role of rad and dsk in spindle function. the degradation of kre and yll030c requires rad23/dsk2 as reasoned above, accumulation of a large amount of substrates can lead to toxicity in cells defective in proteolysis. we examined the stability of the proteins encoded by the eleven isolated genes in wild-type or mutant cells by an expression shut-off assay. two proteins, kre and yll030c are rapidly degraded in wild-type cells, but significantly stabilized in cells lacking rad and dsk, suggesting that they are degraded by the rad23/dsk pathway. deletion of rad and dsk does not significantly alter the half-life of the other nine proteins identified by our screen. previously, we demonstrated that rad works with two different co-factors ufd and png to degrade distinct substrates. here, we examined whether kre or yll030c turn-over requires ufd or png deletion of ufd does not alter the degradation of kre or yll030c. the png1-rad complex is involved in degrading glycosylated proteins. we found that neither protein is glycosylated, suggesting that kre and yll030c are not png substrates. it is likely that these substrates would require co-factors other than ufd and png for their degradation. since these substrates are tagged with gst moiety at their n-terminus, one concern of our results is that the degradation of these proteins may be altered by the gst fusion. to this end, we obtained the morf plasmids expressing yll030c and kre which are tagged at their carboxyl-terminus with both his and the igg-binding site from protein a that theoretically could also affect protein degradation. as shown in figure yll030c and kre are still degraded in rad23/dsk2-dependent manner, suggesting that the involvements of rad and dsk in kre and yll030c degradation are not due to artifacts caused by gst tags. overexpression of these morf plasmids also led to growth retardation in rad23Δ dsk2Δ mutant cells. kre and yll030c are ubiquitylated, proteasomal substrates are these newly identified rad23/dsk substrates destroyed by the proteasome or some other proteolytic activity? to assess the involvement of the proteasome in the degradation of kre or yll030c, we carried out a simple proteasome inhibition assay. we found that the degradation of kre or yll030c is impaired upon mg treatment, suggesting that these proteins are degraded by the proteasome. most, but not all, proteasomal substrates are ubiquitylated. the function of rad23/dsk in proteolysis requires their uba domains, which bind to ub. to determine whether the newly identified substrates are ubiquitylated, we co-transformed the plasmid expressing the gst- and his6-tagged substrate with the plasmid bearing ha-tagged ub into wild-type or rad23Δ dsk2Δ mutant cells. gst-tagged substrates were immunoprecipitated from cellular lysates with gst beads, and processed for western blotting with anti-ha antibody to detect ubiquitylated substrate. we found that both kre and yll030c are ubiquitylated. consistent with the function of rad and dsk at a pre-proteasome but post-ubiquitylation step, much more ubiquitylated species were detected in the rad23Δ dsk2Δ mutants than in wild type cells. the involvement of specific ub receptor for the degradation of kre or yll030c ub-binding proteins that function in proteasome-mediated proteolysis include three uba/ubl proteins, two proteasome-associated factors rpn and rpn and the ufd1-cdc48-npl complex. these ub-receptors have different substrates and sometimes carry out overlapping functions. we examined the stability of gst-tagged kre and yll030c in yeast cells lacking individual ub receptor. both dsk and ufd are required for efficient degradation of yll030c. interestingly, dsk is the only ub receptor involved in kre degradation. furthermore, consistent with the restriction of dsk by rpn, kre and yll030c are degraded faster in rpn10Δ cells than in wild-type cells. kre overexpression suppresses dsk2-induced toxicity to our knowledge, kre is the first substrate that requires only dsk but none of the other ub receptors for its degradation. supporting the direct link between dsk and kre we detected the dsk2-kre interaction by co-immunoprecipitations. it will be important to determine the specific domains of dsk involved in kre22-binding and degradation, and the role of ubiquitylation in the dsk2-kre interaction. since little is known about dsk2-specific substrates, we examined the ub linkage assembled onto kre lys48- and lys63-linked chains were recently linked to the dsk pathway. specifically, we employed ub mutants that had either lys or lys replaced with arg, which in turn blocked ub conjugation to these lysine residues. we found that kre ubiquitylation was abolished by lys48arg mutation, suggesting that the ub chain attached to kre contained lys linkage. among ub-binding proteins involved in proteasome-mediated proteolysis, dsk has the unique property that its overexpression caused toxicity. we examined whether the deletion or overexpression of kre could counteract overdose of dsk. interestingly, increased expression of kre partially suppressed dsk2-induced toxicity. we then asked whether other proteasomal substrates could keep dsk in check. we employed rta, a rad but not dsk substrate, and ubv76-val-βgal that is regulated by both rad and dsk interestingly, the suppression of dsk is substrate specific since the overexpression of rta or ubv76-val-βgal could not overcome dsk2-triggered toxicity. yll030c overexpression also partially suppressed dsk2-induced growth retardation discussion we describe herein the use of a large-scale sdl screen as a new approach to identify proteolytic targets of the rad23/dsk2-dependent pathway. although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging. for example, the first genetically defined ub-dependent proteolytic pathway was discovered in the mid-1980s. despite over a decade of intense efforts, the first cellular substrate of the n-end rule pathway was identified fortuitously. a key cell cycle protein, scc is cleaved at the metaphase-anaphase transition and the resulting carboxyl-terminal fragment is destroyed by the n-end rule pathway to maintain chromosome stability. interestingly, overexpression of scc leads to lethality in mutant cells lacking the n-end rule e enzyme. this finding highlights the difficulty in obtaining proteolytic substrates and also suggests that an sdl screen could be effective for substrate identification. an sdl approach was first employed to uncover genetic interactions in dna replication or chromosome segregation. more recently, it was demonstrated that sdl screens could be used to identify novel targets of a protein kinase. our study further extends the effectiveness of the sdl screen to the ub/proteasome system. the advantages of sdl as a screening tool for proteolytic substrate identification include that the assay is performed in vivo, and the protein expression levels are enhanced by the strong promoter. like other approaches for target discovery, sdl screens have limitations. of approximately yeast genes we screened, only two dsk substrates were isolated. several factors may contribute to the high false negative level: 1) redundancy of various substrate delivery pathways. several known dsk2/rad substrates were not recovered in this screen. in the absence of dsk2/rad other ub adaptor molecules may maintain sufficient proteolysis; 2) genes that lead to severe growth defects in wild-type cells with increased dosage are likely to be excluded from such analysis; 3) not all substrates would elicit enhanced fitness defects in proteolysis mutants; 4) the screen is an indirect way for target discovery. large number of false positives, partly due to a low colony size cutoff, may mask some true positives; 5) screening strategy and criteria for target identification may need further refinement. for example, the rapid turnover or absence of particular substrates might be a requirement under very specific environmental or physiological conditions, which were not covered by our standardized screening protocol. nevertheless, given the difficulty involved in substrate isolation, we believe that sdl provides another way to delineate the functional relationship between a substrate and the specific degradation pathway. to our knowledge, gst-kre is the first substrate that requires only dsk but not other ub receptors for degradation. interestingly, overexpressed kre but not other proteasomal substrates suppresses dsk2-induced toxicity. although the precise mechanism underlying the dsk2-triggered toxic effect is not clear, one possible explanation is that excessive dsk may engage in the abnormal activities that interfere with cell growth. and increased amount of specific substrates may keep dsk in check. our results suggest that kre could serve as an important tool to understand the dsk2-dependent proteolysis. most proteins identified are not direct proteolytic substrates of rad and dsk this is not unexpected since other genes that function in the linear pathway of rad23/dsk target may also induce toxicity in rad dsk mutant cells upon their overexpression. for example, in a separate study on the function of rad and dsk we identified a cell cycle protein involved in the spindle pole body duplication as a degradation target of rad and dsk. interestingly, this protein is known to act upstream of spc a component of the spb. it is likely that increased levels of spc mimic the accumulation of this rad23/dsk target involved in spindle regulation to affect spb duplication, which in turn triggers growth retardation in rad dsk mutant cells. therefore, although the other eight proteins identified by our screen are not regulated directly by rad and dsk we suspect that these proteins work with rad23/dsk targets in vivo and cellular responses to their overexpression require the function of rad and dsk.  in conclusion, as proof-of-concept, we show that an sdl screen offers an effective, complementary method to elucidating biological functions of proteolytic pathways. dsk was first identified years ago in a genetic screen for genes involved in spb duplication. to our knowledge, the spc98-induced toxicity in rad dsk mutant cells is the second piece of evidence linking dsk and rad to spb duplication, which will help us to unravel the specific role of rad and dsk in this process. it is also conceivable that the sdl screen can be adapted to identify relevant ubiquitylation enzymes involved in substrate turnover. the creation of rnai knock-down libraries and orf overexpression collections would extend the use of sdl screens in the ub/proteasome system to higher eukaryotes. abbreviations spb: spindle pole body; ub: ubiquitin; uba: ubiquitin-associated; ubl: ubiquitin-like; sdl: synthetic dosage lethality; his6: hexahistidine; gst: glutathione stransferase. authors' contributions cl, dvd and yl performed the experiments. ba and hr conceived the study and designed the experiments. cl, dvd, ba and hr wrote the manuscript. 
a key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging. in conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways. two proteins identified by the screen are shown to be proteolytic substrates of dsk validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway. using a genome-wide functional screen, we have identified yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins rad and dsk our results suggest that proper functioning of rad and dsk is required for efficient pheromone response, transcription, amino acid metabolism, and dna damage response. however, the specific role of these ubiquitin receptors remains poorly defined.
 ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. a classic role for ubiquitin is to flag proteins for destruction by the proteasome. recent studies indicate that ubiquitin-binding proteins play a pivotal role in transferring ubiquitylated proteins to the proteasome. however, the specific role of these ubiquitin receptors remains poorly defined. a key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.  using a genome-wide functional screen, we have identified yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins rad and dsk our results suggest that proper functioning of rad and dsk is required for efficient pheromone response, transcription, amino acid metabolism, and dna damage response. two proteins identified by the screen are shown to be proteolytic substrates of dsk validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway.  in conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways. 