Lu, Huixin Mazumder, Mostafizur Jaikaran, Anna S. I. Kumar, Anil Leis, Eric K. Xu, Xiuling Altmann, Michael Cochrane, Alan Woolley, G. Andrew A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation 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, <i>Rs</i>LOV, <i>As</i>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 <i>Avena sativa</i>, 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 <i>in vivo</i>, and bind human eIF4E <i>in vitro</i> 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. circularly permuted LOV inhibitor;Eukaryotic Translation Initiation;cLIPS 1 variants;light switchable domain;PYP;study protein synthesis offer;screening optogenetic designs;eIF 4E translation initiation;circularly permuted version;4 EBP 2;eIF 4E;protein synthesis;yeast system;eukaryotic initiation factor 4 E;LOV 2 domain;protein synthesis noninvasively 2019-03-22
    https://acs.figshare.com/articles/journal_contribution/A_Yeast_System_for_Discovering_Optogenetic_Inhibitors_of_Eukaryotic_Translation_Initiation/7955873
10.1021/acssynbio.8b00386.s001