Doctor of Philosophy
Dr. Chris Brandl
Protein biosynthesis is an essential process for all cells. It involves the translation of genetically encoded information into peptides, folding and assembly of peptides into three-dimensional molecules and complexes, and post-translational modification. Molecular chaperones facilitate protein folding so that a native state is achieved. Misfolded proteins and aggregates are toxic within the cell and accumulate due to stress conditions, mutations, and cell aging. Moreover, essential proteins rely on chaperones and co-chaperones for their regulation and activity.
The TTT complex, consisting of Tel2, Tti1, and Tti2, is considered an Hsp90 co-chaperone with specificity for phosphatidylinositol 3-kinase-related kinases (PIKKs). I show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but are sensitive to stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of the PIKK proteins Tra1, Mec1, and Tor1, and affected their localization. I also find that overexpressing Hsp90 or its co-chaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly.
Using a stress sensitive tti2 allele with a Leu to Pro mutation at residue 187, I performed a genetic selection of spontaneous second site mutations. The selection identified a single nucleotide mutation at the same position (C70T) in four tRNAProUGG genes. Since the mutation incorporates the identity element (a G3:U70 base pair) for alanyl-tRNA synthetase into the tRNAPro acceptor stem, I hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. In vitro, tRNAProUGG (C70U) was mis-aminoacylated with alanine by alanyl-tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ~6%. Mistranslating cells expressing tRNAProUGG (C70U) induce a partial heat shock response but grow nearly identically to wild-type.
Hoffman, Kyle S., "Tti2 in PIKK Biosynthesis and Its Use in Identifying Missense Suppressor tRNAs" (2017). Electronic Thesis and Dissertation Repository. 4368.