A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2.
BACKGROUND
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 (e.g. Rad23, Dsk2, Rpn10) 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.


RESULTS
Using a genome-wide functional screen, we have identified 11 yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins Rad23 and Dsk2. Our results suggest that proper functioning of Rad23 and Dsk2 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 Dsk2, validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway.


CONCLUSIONS
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.
Liu
Chang
C
Institute of Biotechnology, Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA. liuc2@uthscsa.edu
van Dyk
Dewald
D
Li
Yue
Y
Andrews
Brenda
B
Rao
Hai
H
D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005804:Genes, Lethal; D016678:Genome; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics* / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C100415:DSK2 protein, S cerevisiae; C490846:RAD23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014452:Ubiquitins; C027208:ubiquinone-binding proteins; D046988:Proteasome Endopeptidase Complex