Using traditional techniques to elucidate the molecular function of an enzyme with multiple roles in many pathways has always been challenging; identifying all the downstream substrates of such enzymes usually requires a systematic approach. The emerging protein chip technology offers a new tool for globally identifying in vitro
substrates of various enzymes. Like other types of large-scale, high-throughput screening (e.g., yeast two-hybrid screening and gene expression profiling), investigators using this approach now face two challenges: how to identify bona fide
, direct in vivo
targets and how to establish a biological connection between a new target and its upstream modulator. Data integration has been proposed as a means of improving the accuracy of the “hits” derived from large-scale screening [27
], but this strategy does not always work when obvious enrichment is lacking. Therefore, careful examination and evaluation of the robustness, reliability, and inherited bias of the proteomic approach is important for identifying the true substrates of an enzyme.
In this study, the use of chip assay allowed us to quickly narrow down the potential substrates from 5,800 to about 100 proteins. By using genetic screening and a less sensitive, solution-based ubiquitination reaction, we were able to rapidly reduce the number of candidates to eight; seven (87.5%) of these were further validated as true substrates of Rsp5 by more rigorous in vivo analyses. This combination of the three methods dramatically improved the probability of identifying bona fide substrates of Rsp5.
Of the yeast strains harboring knockout mutations of the eight in vivo
substrates identified in this study, three (rnr2
Δ, and taf3
Δ) are lethal, two (sla
Δ) are temperature sensitive, and two (rpn10
Δ and nkp2
Δ) show reduced fitness () [28
]. It is intriguing that many downstream targets of Rsp5 are also essential for viability. Although previous studies have suggested that the essential requirement for Rsp5 is related to the oleic acid pathway [29
], our data seem to indicate that the vital importance of Rsp5 is correlated with its effects on several additional essential pathways. On the basis of the known functions of the substrates we have identified, it is likely that Rsp5 plays a pivotal role in a complicated network that regulates various crucial downstream events, including proteasome function, DNA synthesis, chromosome segregation, cytoskeleton assembly/ endocytosis, and transcription (). These results should encourage in-depth studies related to the function of ubiquitin E3 ligase.
Among the 145 reported Rsp5-interacting proteins containing the PXY motif [30
], only five were ubiquitinated in vitro
by Rsp5 on the protein chip, and two of the five were confirmed as in vivo
substrates of Rsp5. This situation may be explained by the notion that a significant portion of these proteins acts as adaptors for Rsp5. Emerging evidence suggests that many Rsp5-interacting proteins recruit Rsp5 to particular subcellular compartments to facilitate the ubiquitination of their substrates [31
]. Moreover, the WW domains of Rsp5 may interact only with phosphorylated PXY motifs, and some adaptor proteins may mediate substrate interactions from which proline-rich PXY motifs are absent [32
]. Therefore, it would be useful to carry out the Rsp5 ubiquitination on protein chips in the presence of an adaptor protein or after pre-treatment with specific kinases.
Previous studies have identified 11 proteins as bona fide
substrates of Rsp5, as determined by Rsp5-dependent ubiquitination in vivo
]. Most of these substrates either have a low protein abundance on chips because they are membrane proteins and are therefore difficult to express and purify (Fur4, Gap1, Lsb1, Sna4, and Ydl203c), or because they have proved to be unstable in separate attempts at purification (Rpb1 and Ste2). For the rest (Rvs167, Mga2, Sna3, and Ydl203c), we observed only moderate ubiquitin signals for Rvs167 and Sna3, suggesting that the protein chip approach has its own bias against certain proteins. Moreover, Gupta et al.
used a similar proteome-wide approach but found a different spectrum of substrates (Fig. S5
]. The discrepancy can conceivably be explained by the different strategies used to validate the candidates. In our study, we found that combining the result of on-chip biochemical experiments with genetic interaction profiling significantly increased the probability of identifying biologically relevant substrates (). Our results further suggest that protein-protein interaction is not required for substrate identification, since only four of the eight validated substrates have been previously shown to interact with Rsp5.
Among the validated in vivo
substrates of Rsp5, Rnr2 is of particular interest. Rnr2 is a highly conserved ribonucleotide reductase (RNR) that coverts nucleotides to deoxynucleotides in a reaction dependent on a diferric-tyrosyl cofactor [34
]. A heterozygous null mutant of RNR2
is associated with hypersensitivity to DNA damage and to treatment with HU, a chemical inhibitor of the RNRs [19
]. After DNA damage, Rnr4 is redistributed within cells, perhaps reflecting an as yet-unidentified posttranslational mechanism [35
]. Our results suggest that Rnr2 localization is determined by Rsp5 activity as well as HU treatment. Rnr2 was found in both the nucleus and the cytoplasm in the WT strain, but the majority of the Rnr2 was localized to the nucleus in the rsp5
mutant. The fact that the RNR complex needs to be present in the cytoplasm in order to be functional [34
] may help explain why the rsp5
mutant is hypersensitive to HU at the semi-permissive temperature.
We conclude that a combination of proteome microarray-based biochemical assays and genetic interaction screens offers a powerful platform for identifying bona fide substrates of enzymes involved in various cellular pathways and our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.