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A bacterial genetic selection system for ubiquitylation cascade discovery

Nature Methods volume 13, pages 945–952 (2016)Cite this article

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Abstract

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.

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Figure 1: Bacterial genetics approach for selection of ubiquitylated proteins.
Figure 2: Identification and characterization of NleG6-3 as E3 ligase.
Figure 3: The bacterial selection system facilitates the identification and characterization of Ub receptors.
Figure 4: Structural insight into a predicted ENTH–Ub interface.
Figure 5: Characterization of the ENTH–Ub binding interface.
Figure 6: Sem1 is a ubiquitylation substrate of Rsp5.

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Acknowledgements

This manuscript is dedicated to the memory of Amos Oppenheim (1934–2006), my Ph.D. mentor and friend who was influential in determining this choice of research. We thank the teams of ID29 and ID14-4 beamlines at ESRF for their exceptional help and for maintaining and upgrading the facility. We thank S.D. Emr and H.C. Ho (Cornell University, Ithaca), A. Chitnis and S.Y. Choi (NIH, Maryland) for providing the rsp5-1 strain and Epn1 cDNA, respectively, and for fruitful discussions. We thank N. Balaban (The Hebrew University, Jerusalem), Z. Ronai (SBP at La Jolla, California), J. Bonifacino (NIH, Maryland) and J. Hurley (UC Berkeley) for providing the pZE21 vector, SIAH2, GGA1/2 and STAM1-VHS clones, respectively. We thank I. Rosenshine (The Hebrew University, Jerusalem) for providing EHEC gDNA and for influential discussions. This work was supported by grants from the Israeli Science Foundation (no. 464/2011) and the Israeli Ministry of Health (no. 3000005108) to G.P.

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Authors and Affiliations

  1. Departments of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Olga Levin-Kravets, Neta Tanner, Noa Shohat, Ilan Attali, Tal Keren-Kaplan, Anna Shusterman, Shay Artzi, Yael Reshef, Xiaojing Shi, Ori Zucker, Tamir Baram, Corine Katina, Inbar Pilzer & Gali Prag

  2. Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

    Alexander Varvak & Shay Ben-Aroya

  3. Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

    Gali Prag

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  1. Olga Levin-Kravets
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Contributions

O.L.-K. constructed and evaluated the selection system, constructed the yeast E2 library, determined the structure of zebrafish ENTH, performed genetic analysis of ENTH–Ub interactions and wrote the manuscript; N.T. crystalized and determined the structure of yeast ENTH domain and performed yeast ENTH–Ub binding assays; N.S. identified and characterized NleG6-3 and inhibition assays; S.A. performed inhibition assay with SIAH2; A.V. performed SPR measurements; Y.R. performed ubiquitylation assays with zebrafish Epn1; X.S. performed the genetic analysis of GGA and STAM; I.A. assisted the SPR and growth-data analyses; T.K.-K., A.S., O.Z. and T.B. performed some selection experiments and analysis; C.K. cloned the HRS/STAM to the genetic system; I.P. provided technical help; S.B.-A. helped with library construction; G.P. conceived the idea, designed the experiments, supported structures determination and data analyses and wrote the manuscript.

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Correspondence to Gali Prag.

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Competing interests

The technology transfer company of Tel Aviv University (RAMOT) has filed a provisional based on our developed system. The provisional number is 62/371,881.

Integrated supplementary information

Supplementary Figure 1 Schemes show two major hurdles in the ubiquitin field.

The two pose challenges in assigning specific associations of components along ubiquitin cascades. (a) Ubiquitylation is rapidly reversed by deubiquitylases. In human there are about hundred deubiquitylating enzymes (DUBs) which efficiently and rapidly remove the ubiquitin moiety from targeted proteins. (b) Ubiquitylation cascades are multiplex. For example eight different substrates were found for the BRCA1 E3-ligase. Similarly, 7 different E3-ligases were demonstrated to ubiquitylate p53.

Supplementary Figure 2 Architecture and sequence of the linkers connecting the DHFR fragments to ubiquitin and to ubiquitylation target

Schematic illustration of the constructs used in the selection system. pND-Ub vectors contain a N-Terminal DHFR fragment fused through the flexible linker (linker 1) to Ub. In the pCD-Sub the C-Terminal fragment of the DHFR was fused through the flexible linker (linker 2) to the His6-MBP-substrate or directly to the substrate.

Supplementary Figure 3 Domain architecture of membrane associated Ub-receptors.

Ub moieties mark the binding sites (orange circled U’s).

Supplementary Figure 4 Ent1-ENTH domain directly binds ubiquitin.

(a) Shows crosslinking assay of Ent1-ENTH domain with increased concentrations of Ub. A mild crosslinker, disuccinimidyl suberate (DSS) was used. Reactions were resolved by SDS-PAGE and Coomassie blue stained. (b) Shows crosslinking assay (like in a) of Ent1-ENTH domain with wild-type and Ub I44E mutant for various incubation times (as indicated). (c) Scheme of yeast and zebrafish Epsin-1 derivative constructs show blow in (d) and (e). (d,e) Ubiquitylation of yeast and zebrafish Epsin-1 derivatives. His6-Ub was co-expressed with E1 and Ubc4 along with GST-Epsin1 derivatives. Proteins were purified on GSH-beads and ubiquitylation was detected by western blot using anti-Histag antibody as described in17.

Supplementary Figure 5 Structural divergence within the ENTH domains.

a. The coordinates of several ENTH domains including yeast (magenta), zebrafish (white), and three structures of rat ENTH domains (cyan, red and yellow), were superposed. The axis of helix-8 from each of the structures were calculated. Then the angles among the derived helices were compared. b. Structural differences between the loops tethering helices number 3 and 4 in yeast and zebrafish ENTH domains are presented.

Supplementary Figure 6 ENTH/VHS domains can simultaneously associate with membranes, M6PR-tail and ubiquitin.

Superimposing the ENTH complex with the lipid phosphatidylinositol-4,5-bisphosphate, GGA3-VHS complex with the Mannose-6P Receptor tail and the STAM1-VHS:Ub shows that membrane and Ub associations occur at opposite sides of the domain and therefore can occur simultaneously.

Supplementary Figure 7 Surface Plasmon Response (SPR) analysis of ENTH:Ub interaction.

Crude response curves for the wild-type and indicated mutants are shown for each of the measured analyte concentrations

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1 and 2. (PDF 1385 kb)

Supplementary Table 3

Supplementary Table 3 (XLSX 11 kb)

Bacterial growth time-lapse shows Rsp5-dependent Sem1 ubiquitylation

Time-lapse scans showing ubiquitylation dependent bacterial growth assay. Selective (left) and non-selective (right) plates were spotted with the indicated bacteria: DHFR (positive control); complete system contains E1-E2-E3 and Sem1; ΔRsp5; ΔRsp5,ΔUbc4; ΔUb. Scans were taken every 30 minutes for 100 hours (time shown at top left). (GIF 61150 kb)

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Levin-Kravets, O., Tanner, N., Shohat, N. et al. A bacterial genetic selection system for ubiquitylation cascade discovery. Nat Methods 13, 945–952 (2016). https://doi.org/10.1038/nmeth.4003

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