A bacterial genetic selection system for ubiquitylation cascade discovery.
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Olga
O
Levin-Kravets
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Neta
N
Tanner
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Noa
N
Shohat
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Ilan
I
Attali
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Tal
T
Keren-Kaplan
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Anna
A
Shusterman
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Shay
S
Artzi
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Alexander
A
Varvak
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Yael
Y
Reshef
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Xiaojing
X
Shi
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Ori
O
Zucker
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Tamir
T
Baram
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Corine
C
Katina
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Inbar
I
Pilzer
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Shay
S
Ben-Aroya
Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.|Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.
Gali
http://orcid.org/0000-0003-4703-7161
G
Prag
D000072017:Deubiquitinating Enzymes / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D008958:Models, Molecular; D010957:Plasmids; D012641:Selection, Genetic; D015398:Signal Transduction / Q000235:genetics; D013869:Thiolester Hydrolases / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D054875:Ubiquitination / Q000235:genetics*
D029968:Escherichia coli Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C527558:EntH protein, E coli; D013869:Thiolester Hydrolases; D000072017:Deubiquitinating Enzymes
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