1. Effects of Ifh1 mutations on lifespan and Gcn4 expression Ashley L. Evans1,2, Audrey T. Bell1,3, Steven Waite1,4, Dan Schreil1,5, Christine E. Robbins1,4, Bhumil B. Patel1, Ling Cai6, Mark A. McCormick1, Benjamin Tu6, Brian K. Kennedy1,7 1 Buck Institute for Age Research, Novato, CA, 5, 2Cloverdale High School, Cloverdale, CA, 3Marin Academy, San Rafael, CA, 4Sonoma State University, Rohnert Park, CA,5 Dominican University, San Rafael, CA, 6 UT Southwestern, Dallas, TX, 7Aging Research Institute, Guangdong Medical College, Dongguan, Guangdong, P.R.China Ifh1 and Fhl1 transcribe the RP genes whose deletion increases lifespan Introduction Deletions in some yeast ribosomal proteins has been shown to extend replicative lifespan. It turns out that Ifh1 and Fhl1 are essential genes encoding transcription factors that cooperate to transcribe the ribosomal protein genes, whose deletion has been shown to extend RLS. Gcn4 is a transcription factor that responds to amino acid starvation. Lifespan-extending ribosomal protein deletions show increased translation of Gcn4. We have created mutant versions of the essential IFH1 gene that alter Ifh1’s phosphorlyation. We found that some of these affected lifespan and Gcn4 levels. Serine to Alanine mutations that prevent phosphorylation of Ifh1 extend yeast lifespan Transcription of ribosomal proteins Replicative aging measures how many times a yeast cell divides before it stops We cannot delete either of these genes, but we created mutant versions of IFH1 that change phosphorylation at serine 969 Mother Cell We can measure Gcn4 levels with our dual-luciferase plasmid Daughter J Vis Exp. 2009 Jun 25;(28). Serine (S)- this is normally there Phospho-serine – this is the modified version Aspartic Acid (D)– one possible replacement amino acid – (appears phosphorylated) Alanine (A) –another possible replacement amino acid (can’t be phosphorylated) Deletion of some yeast ribosomal proteins has been shown to extend replicative lifespan We found an increase in Gcn4 in the serine to alanine mutant Numbers in parentheses are mean lifespans Δ means the gene has been deleted This lifespan extension depends on Gcn4 Serines (S) mutated to alanine (A) cannot be phosphorylated, while serines mutated to aspartic acid (D) may mimic phosphorylation Acknowledgements Thanks to the Buck Institute, the Summer Scholars program, Julie Mangada, and Mark McCormick, Brian Kennedy, and the Kennedy Lab, who have made this experience possible.