A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation.

Lu, Huixin; Mazumder, Mostafizur; Jaikaran, Anna S I; Kumar, Anil; Leis, Eric K; Xu, Xiuling; Altmann, Michael; Cochrane, Alan; Woolley, G Andrew (2019). A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation. ACS synthetic biology, 8(4), pp. 744-757. American Chemical Society 10.1021/acssynbio.8b00386

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The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development, and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis noninvasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, AsLOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photoactivated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate human eIF4E-dependent translation initiation in a mechanistically defined manner.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Altmann, Michael

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

2161-5063

Publisher:

American Chemical Society

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

26 Sep 2019 09:25

Last Modified:

03 Nov 2019 00:44

Publisher DOI:

10.1021/acssynbio.8b00386

PubMed ID:

30901519

Uncontrolled Keywords:

circular permutation genetically encoded optogenetics photocontrol photoisomerization translation

BORIS DOI:

10.7892/boris.133479

URI:

https://boris.unibe.ch/id/eprint/133479

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