A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation

Abstract

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, LOV, 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 photo-activated 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 of human eIF4E-dependent translation initiation in a mechanistically defined manner.

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

“…[129] Live-cell readout, such as yeast cell growth, could be repurposed to validate interactions between photoactivatable proteins, particularly dimerizers. [122,130] An alternative way is to use biosensors that respond to the activation of photoactivatable proteins to confirm the system's functionality. For instance, the dimer dependent red fluorescence protein (ddFP) was engineered only to emit fluorescence when the synthetic G protein receptor is appropriately activated by light.…”

“…INT+: Positive interaction; INT−, negative interaction; ddFP, dimerization‐dependent fluorescence protein c) Efficient resources for conducting an experiment or collecting data. References of each strategy are listed as follows: machine learning directed evolution, [ 119,120 ] interface directed mutagenesis, [ 122 ] computational prediction, [ 31,123,124 ] native mass spectrometry, [ 128 ] SEC‐HPLC, [ 129 ] yeast screening, [ 122,130 ] signaling biosensor, [ 131 ] optoplate, [ 132 ] LAVA plate, [ 133 ] optobase, [ 1a ] and the optogenetic resource center. [ 1 ] The image of the optoplate is reproduced with permission.…”

The rise and shine of yeast optogenetics

“…(g) An optogenetic switch based on the interaction of the UV-light photoreceptor UVR8 with the COP1 protein. The optogenetic switch architecture is also based on the Y2H system, similar to the switch shown in panel (a) chromatin modification, and heterotrimeric G-protein activation (Garcia-Marcos et al, 2020;Lerner et al, 2018;Lu et al, 2019;Meriesh et al, 2020;Spiltoir et al, 2016;Yumerefendi et al, 2016;Yumerefendi et al, 2018). Interestingly, a modified version of the improved light-induced dimer (iLID) system (Guntas et al, 2015), which is also based on the AsLOV2 photoreceptor, led to the development of the 'Corelets' systems for protein clustering in multiple biological platforms, including yeast (Bracha et al, 2018).…”

A light way for nuclear cell biologists

“…mRNA translation has been so far controlled in four ways: by controlling translation initiation using light to (i) recruit eukaryotic initiation factor 4E (eIF4E) to the mRNA of interest ( 115 , 116 ) or (ii) release inhibition of eIF4E using a light-inducible form of an eIF4E binding protein (opto-4EBP) ( 117 ); (iii) by interfering with translation recruiting to an aptamer sequence placed between the mRNA CAP and the Kozak sequence a light-inducible RNA binding protein called PAL ( Fig. 1C ) ( 118 ) and (iv) by sequestering with light the target mRNA into protein clusters, which makes them less accessible to the ribosomes ( 119 ).…”