Ballabio had become interested in the lysosome while studying a particular kind of lysosomal storage disease. Multiple sulfatase deficiency causes scaly skin, stiff joints, seizures and developmental delays. The symptoms arise from mutations in a gene that, as Ballabio’s group discovered, is essential for the activation of a group of enzymes called sulfatases, many of which are lysosomal.

That discovery by Ballabio’s team, along with other studies of rare lysosomal disorders, convinced Ballabio that cells must have a system to boost lysosomal activity and a way to start making more lysosomes as cellular trash piles up. To do this, “you need to control the function of many different genes,” Ballabio said. He set out to find the master regulators that do this.

In 2009, his team reported that it had found an important one. They called it “transcription factor EB,” or TFEB. In the cell’s nucleus, TFEB binds to DNA sequences in many lysosomal genes and controls the rate at which they make proteins.

Precisely how TFEB’s activity could reflect the cell’s needs for lysosomes so comprehensively, however, was still unknown. But an answer would soon emerge from work that, at least initially, had nothing to do with lysosomes.

A Seat of Signaling

When Roberto Zoncu arrived as a postdoc at Sabatini’s lab at the Whitehead Institute in 2008, lysosomes were not uppermost in his mind. The lab’s focus was (and in many ways still is) on the enzyme that Sabatini had discovered in mammalian cells in 1994 and dubbed mechanistic target of rapamycin (mTOR). Implicated in aging and a slew of diseases including cancer and diabetes, mTOR signals cells to grow and divide under a surprisingly wide variety of circumstances. “One of the big motivating questions for us has been: How does that happen?” Sabatini said. “How does mTOR manage to sense so many things, integrate those signals and drive growth?”

A critical clue came when the team tracked the protein’s movements within cells. When cells were bathed in amino acid-free media, mTOR seemed to spread evenly throughout the cytoplasm. But if the media contained amino acids, within minutes mTOR moved into distinct clusters at specific locations inside the cell, shepherded there by other proteins called Rag GTPases. The enzymatic activity of mTOR depended on its reaching those locations, but the proteins that guided it there did not appear to turn it on. “We were stuck,” Sabatini said.

Zoncu therefore set out to learn what was special about where the mTOR protein was going in response to amino acids. In a key experiment, he stained cells with pairs of fluorescent antibodies: a red one that bound mTOR and a green one designed to bind to a protein associated with a different organelle in each round of the experiment. He then examined the cells under the microscope, looking for where the green and red fluorescent tags overlapped. This would indicate what else was located in the spots where the mTOR clustered.

Scanning the slide that stained for mitochondria — a potential target of huge metabolic importance — Zoncu found no overlap. He moved on to the slide for the next organelle, and the next. Still no overlap. “I almost lost hope,” Zoncu recalled.

Then came the lysosome slide. “All of a sudden, everything matched perfectly,” he said. The red mTOR staining and the green staining for lysosomal marker LAMP2 overlapped 100 percent.

Revving Up the Recycler

Those results added further support to the data Sabatini reported at the Maine conference in 2008 to his underwhelmed audience. But even Zoncu acknowledges that skepticism might have been warranted. Lysosomes, he says, could still “have just been a landing pad” — a convenient place for mTOR to touch down during activation.

Yet later experiments suggested otherwise. When Zoncu extracted lysosomes from cells and loaded them with amino acids, he saw that the more amino acids they carried, the more mTOR clustered on their surface and became active. (The enzyme mTOR forms two protein complexes in the cell; mTOR complex 1 [mTORC1] is the one found on lysosomes.) Those experiments, published in 2011, show that mTORC1 responds to the lysosomal contents, Sabatini says — as though the lysosomes tell mTORC1 about the amino acids they hold and mTORC1 adjusts its behavior accordingly.

Widening the Cellular Conversation

When Ballabio’s lab and Sabatini’s learned of one another’s results and joined forces, they soon worked out how the mTORC1 and TFEB pieces of the puzzle fit together, publishing the solution in 2012. In a healthy, well-fed cell, lysosomes have a cornucopia of proteins to break down to their amino acid components, and those amino acids work with proteins on the lysosome surface to anchor mTORC1 and activate it. The mTORC1 in turn keeps cytoplasmic TFEB out of the nucleus. When a cell becomes starved or stressed, mTORC1 drops away from the lysosome and TFEB is freed to bind its targets on the nuclear DNA. Acting as a master sensor of lysosomal function, TFEB turns on genes for more lysosomal enzymes.