Biased regulation of protein synthesis and hypoxic death by a conditional raptor mutation

Abstract

Mechanistic target of rapamycin (mTOR) functions in mTOR complex 1 (mTORC1) with raptor to match metazoan metabolism to available nutrients to regulate multiple cellular, physiological, and pathological processes. Hypoxic cellular injury is influenced by the mTORC1 pathway, but whether its activity promotes or prevents injury is unclear, and which mTORC1-regulated mechanisms control hypoxic injury are obscure. Here, we report the discovery of a hypoxia-resistant, temperature-sensitive raptor mutant in an unbiased forward mutagenesis screen in C. elegans. This raptor mutant is both hypoxia resistant and long lived at intermediate temperatures, while unable to develop at higher temperatures. Temperature-shift experiments show that the conditional hypoxia resistance can be induced in the raptor mutant immediately prior to the hypoxic insult. At these intermediate temperatures, the raptor mutation selectively reduces protein synthesis without affecting autophagy, and epistasis experiments implicate mTOR-targeted translation regulators as components of the hypoxia resistance mechanism. Using the conditional developmental arrest phenotype in a selection for suppressors of raptor loss of function, we isolated multiple second-site raptor missense mutants, whose mutated residue is predicted to interact with RagA, a raptor-binding protein. These suppressor mutations restore normal protein synthesis, hypoxic sensitivity, and lifespan and thereby implicate raptor-RagA interactions as critical to these biological processes.

Keywords: C. elegans; anoxia; autophagy; cell death; conditional mutant; mTOR; mTORC1; metabolism; rapamycin; translation.