The Interaction Between eIF2B and YBR159W

1. YBR159W, A Fatty Acid Synthesis Enzyme, Targets eIF2B to the ER Membrane and Increases the Efficiency of ER Protein Translation Christopher M. Browne, Parimal Samir, Connie Weaver, Andrew J. LinkVanderbilt University School of Medicine, Nashville, TN, USA Results Abstract Results The Interaction Between eIF2B and YBR159W Using mass spectrometry-based proteomics, I have discovered a novel protein-protein interaction in S. cerevisiae between eIF2B and the ER membrane bound fatty acid biosynthesis enzyme YBR159W. YBR159W is the beta-ketoacylreductaserequired for very-long-chain fatty acid (VLCFA) elongation. Till now, a direct link between translation initiation and fatty acid biosynthesis has not been shown. My data suggests that the interaction helps to increase the rate of protein translation on the rough ER. Deletion of YBR159W Leads to Disruption of eIF2B Bodies Background Hierarchical clustering of the MS/MS analysis of Tandem Affinity Purifications (TAP) of various yeast proteins shows that all five subunits of eIF2B interact with YBR159W. WT WT GCD7 GCD7 • eIF2B is an essential guanine nucleotide exchange factor (GEF). • Only known target is eIF2 • Four of five yeast subunits are essential • Cytosolic localization • YBR159W is required for VLCFA synthesis. Deletion causes: • slow growth and temperature sensitivity. • Loss of fatty acids 24 and 26 carbons in length • Changes in lipid profile • YBR159W is ER membrane bound eIF2B normally localizes to a small number of distinct foci in the cell. In the ybr159wΔ background these foci become many smaller punctate structures. ybr159wΔ Interaction map of eIF2B and YBR159W Polysome Profiling Deletion of YBR159W causes a very slow growth phenotype Yeast 2-hybrid analysis further confirms the interaction between eIF2B and YBR159W YBR159W Increases the Efficiency of Membrane Associated Protein Translation WT PI 26:0 18:1 PI 26:0 20:1 The reduced ratio of membrane protein synthesis to cytoplasmic protein synthesis Indicates a defect in membrane protein translation YBR159W-degron 2hr. depletion The slow growth rate of ybrs159wΔaccounts for the strain’s disturbed polysome profile. The ybr159wΔ strain has a large amount of non-translating 80S ribosomes and low polysome levels. lip1Δ is also a slow growing strain and produces similar polysome profiles. LC-MS Lipid Profile of WT and a conditional YBR159W-degronstrain after 2hrs of YBR159W depletionshowing reductions in very-long-chain fatty acids. Hypothesis Conclusions A locally increased concentration of eIF2B at the ER membrane would aid in the rapid turnover of translation complexes needed for membrane bound and secreted protein translation. • The interaction between YBR159W and eIF2B is distinct from either proteins’ known functions. • YBR159W appears to target eIF2B to the ER membrane to increase its local concentration and allow for more efficient, localized translation initiation. Deletion of YBR159W causes an overall reduction in protein translation Wildtype levels of GCN4 de-repression indicate no defects in the ybr159wΔ strain’s efficiency of translation initiation This work was funded via: NIH 5R01GM064779-09