Regulation of the Target of Rapamycin Signaling Pathway in Saccharomyces cerevisiae

<p>An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and <em>in vivo</em> protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, <em>Saccharomyces cerevisiae</em>. Mutations in <em>SAC7</em> and <em>FAR11</em> suppressed lethality of both <em>lst8</em> and <em>tor2-21</em> mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2.</p> <p>Far11, a component of a six-member complex, was found to interact with Tpd3 and Pph21, conserved components of Protein Phosphatase 2A (PP2A) via co-immunoprecipitation. Mutations in <em>FAR11</em> and <em>RTS1,</em> which<em> </em>encodes a PP2A regulatory B subunit, restore phosphorylation to the TORC2 substrate Slm1 in a <em>tor2-21</em> mutant. These data suggest that TORC2 signaling is antagonized by Far11-dependent PP2A activity.</p> <p>To characterize the assembly of the Far complex <em>in vivo</em>, intracellular localization of the Far complex was examined by fluorescence microscopy. It was found that the Far complex localizes to the endoplasmic reticulum (ER). The data show that Far9 and Far10 are tail-anchored proteins that localize to the ER first and recruit a Far8-Far7-Far3 pre-complex. Far11 is found at the ER only when all other Far proteins are assembled at the ER. Surprisingly, ER localization is required for the Far Complex’s role TORC2 signaling because deletion of the tail-anchor domain of Far9 results in partial bypass of the <em>tor2-21</em> mutant growth defect at 37 ˚C.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation-Restricted

Discipline thesis:degree_discipline

Chemistry

Year

2013

Author dc:creator

• Pracheil, Tammy

Contributors dc:contributor

• Liu, Zhengchang
• Schluchter, Wendy
• Rees, Bernard

• Lst8
• Far11
• yeast
• protein phosphatase 2A
• rapamycin
• TOR signaling
• Biochemistry
• Biology
• Biotechnology
• Molecular Biology

Repository record dc:identifier

https://scholarworks.uno.edu/td/1662

OAI identifier oai:identifier

oai:scholarworks.uno.edu:td-2647