sb8b00386_si_001.pdf (791.75 kB)
A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation
journal contribution
posted on 2019-03-22, 00:00 authored by Huixin Lu, Mostafizur Mazumder, Anna S. I. Jaikaran, Anil Kumar, Eric K. Leis, Xiuling Xu, Michael Altmann, Alan Cochrane, G. Andrew WoolleyThe 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.
History
Usage metrics
Categories
Keywords
circularly permuted LOV inhibitorEukaryotic Translation InitiationcLIPS 1 variantslight switchable domainPYPstudy protein synthesis offerscreening optogenetic designseIF 4E translation initiationcircularly permuted version4 EBP 2eIF 4Eprotein synthesisyeast systemeukaryotic initiation factor 4 ELOV 2 domainprotein synthesis noninvasively
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC