Background:Rictor is an essential component of the mammalian target of rapamycin complex 2 (mTORC2). Results: Rictor contains two central regions that (i) bind mSin1 and LST8 and (ii) function in multisite acetylation. Conclusion: Rictor acetylation is a post-translational modification that potentiates mTORC2 activity. Significance: Understanding the molecular mechanisms by which acetylation potentiates mTORC2 activity links nutrient signaling with critical metabolic kinases.

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N-and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regiospecific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

The Ser/Thr protein kinase mTOR is a critical regulator of cell growth, survival and metabolism in response to growth factors. mTOR functions in at least two distinct multiprotein complexes known as mTOR complex (mTORC)-1 and-2. mTORC2 phosphorylates Akt at S473, an event required for increased Akt kinase activity. Although it is known that the mTORC2 components Rictor and mSin1 are required for the stability and activity of mTORC2, little is known about post-translational modifications that regulate mTORC2. Work presented in this thesis identifies Rictor acetylation as a positive regulator of mTORC2-mediated phosphorylation of Akt at S473. Inhibition of deacetylases, including the NAD +dependent Sirtuins, promotes Rictor acetylation and IGF1-mediated Akt phosphorylation at S473. While mapping the major acetylated regions contained within Rictor we identified two important regions: 1) a region critical for interaction with mSin1.1 and LST8 that subsequently promotes mTORC2 stabilization, and 2) an adjacent acetylated region localized between amino acids 1041 and 1137. Analysis of this acetylated region in Rictor identified 9 lysines that could potentially be post-translationally modified, each of which we divided into 4 distinct groups or modules (M1-M4). Site-directed mutagenesis (lysine to arginine) of each of the lysines located within two of the modules (M2 and M4) reduced Rictor acetylation and IGF1-dependent mTORC2 kinase activity. These results indicate that multiple-site acetylation of Rictor signals for increased activation of mTORC2 in response to IGF1 stimulation. To further examine