Journal News

Flipping the switch

Courtney Chandler
Nov. 23, 2021

Eukaryotic cells must balance multiple factors before they can grow. The mechanistic target of rapamycin complex 1, or mTORC1, pathway senses and responds to various environmental cues, including nutrient availability, to control protein synthesis and cell growth.

The RagA–RagC GTPase heterodimer is required to activate mTORC1. Depending on amino acid availability, the RagA and RagC subunits will be oppositely loaded with either guanosine triphosphate or guanosine diphosphate, known as GTP and GDP. This variable nucleotide binding induces conformational changes in the heterodimer structure that promote the interaction or release of mTORC1.

When concentrations of amino acids in the cell are high, RagA is bound to GTP, and RagC is bound to GDP — this structural conformation facilitates mTORC1 interaction and activation of protein synthesis at the lysosomal surface. The reverse GTP/GDP loading leads to inhibition of mTORC1 and release from the lysosomal surface into the cytosol.

Until recently, researchers did not understand clearly how RagA and RagC maintain their oppositely nucleotide-loaded state to regulate mTORC1. In an article in the Journal of Biological Chemistry, a team from the University of Massachusetts Chan Medical School describes the molecular mechanism behind this regulation and the consequences of disrupting this balance.

Kuang Shen, an assistant professor at UMass and senior author on the article, described the RagA–RagC complex as a complicated light switch that controls mTORC1 — only when one switch is up and one switch is down will the mTORC1 lightbulb turn on.

“We were curious about the mechanism of regulation, because for us it’s like a black box and we didn’t understand how it worked,” Shen said.

Single-hydrogen-bond-890x376.jpg
Kuang Shen & Shawn Egri
A single hydrogen bond (in red in the box on the right) ensures the Rag GTPase complex can switch appropriately between the on and off states to regulate mTORC1 signaling and protein synthesis.

To peer into the black box, graduate student Shawn Egri used enzyme kinetics, structural biology and chemistry tools to probe the mechanism behind the regulation. Egri said he purified between 30 and 40 RagA–RagC mutants with individual amino acid differences and tested their activity one by one.

Eventually, the researchers identified mutants that eliminated normal RagA–RagC activity. They found that one interdomain hydrogen bond is all it takes to keep the switch in one position or the other and prevent it from spontaneously flipping between the two states.

“It was really amazing that just one hydrogen bond has this profound of an impact,” Shen said.

They next introduced mutants in which the interdomain hydrogen bond was broken (meaning the Rag GTPase cannot maintain its oppositely nucleotide-loaded state) into cells. They observed that with the interdomain hydrogen bond abolished, the GTPase subunits could not maintain their original nucleotide-loaded states in cells, leading to distorted responses to amino acid availability and altered mTORC1 interactions.

“It was surprising to us that our results (with purified proteins) translated so strongly in cells,” Egri said. “The phenotype was very clear even in wild-type cell background.”

In the future, Egri and Shen want to lock the complex in the on and off positions to see how that affects interactions with upstream regulators and downstream effectors of the mTORC1 pathway. This could help piece together a better idea of the signals and pathways that regulate the essential processes of protein synthesis and cell growth.

Courtney Chandler

Courtney Chandler is a postdoctoral researcher at the University of Maryland, Baltimore, and an industry careers columnist for ASBMB Today.

Join the ASBMB Today mailing list

Sign up to get updates on articles, interviews and events.

Latest in Science

Science highlights or most popular articles

How a tiny pet store fish became the center of neuroscience research
News

How a tiny pet store fish became the center of neuroscience research

Nov. 27, 2021

The tropical zebrafish is used extensively in genetics, neuroscience and development labs worldwide.

Science is a human endeavor
Essay

Science is a human endeavor

Nov. 26, 2021

The author learned some difficult and important lessons when he decided to pursue errors in a Nobel laureate’s work.

‘Fatty retina’: A root cause of vision loss in diabetes?
Lipid News

‘Fatty retina’: A root cause of vision loss in diabetes?

Nov. 25, 2021

Abnormalities of lipid metabolism are common in diabetes, so the authors reasoned that the retina might switch its programming in response to an abundance of fuel.

From the journals: MCP
Journal News

From the journals: MCP

Nov. 24, 2021

What’s the role of CD151 in triple-negative breast cancer? How similar are nonstructural proteins between coronavirus homologs? What proteins are candidates for targeting oral cancer?

Gain-of-function research: All in the eye of the beholder
News

Gain-of-function research: All in the eye of the beholder

Nov. 21, 2021

Should work done on coronaviruses in Wuhan, China, be considered “gain-of-function” research? Opinions differ.

The music of proteins made audible
News

The music of proteins made audible

Nov. 20, 2021

Many features of proteins are analogous to music. Mapping these features together creates new musical compositions that help researchers learn about proteins.