The inside of a cell is like a house that has a lot of rooms. Little compartments called organelles are the intracellular versions of organs like the liver or the pancreas in the body. Each organelle is assigned its own specialized set of tasks. For example, there is the nucleus, home to the cell’s DNA, the energy-producing mitochondria and the ribosomes that put together whole proteins.
A lesser-known organelle is the humble vacuole, long conceptualized as the cellular wastebasket. Traditionally, vacuoles are known for cleaning up the cell by chopping up and disposing of old or defective proteins. They do this by maintaining an acidic environment in their interior. On a vacuole’s membrane resides an array of protein pumps that move positive hydrogen ions (H+) from the surrounding cytosol into the vacuole, which increases their acidity.
Recent research has shown that vacuoles are more than just a cell’s garbage dump. In fact, they play a vital role in the way the cell integrates information about its nutrient status, and they release or sequester nutrients according to the cell’s needs, which vary throughout the cell cycle.
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Vacuoles use a proton gradient (the difference in acidity between the inside and outside of the membrane) to provide energy for proteins on their surface known as amino acid transporters. The transporters enable the vacuole to move amino acids inside and sequester them—and then release them at a specific point in the cell cycle when a cell is just about to divide to produce a daughter cell.
A team of researchers at Calico Labs in San Francisco, founded by Alphabet, recently showed that to pull this off, the vacuole undergoes dramatic oscillations in pH—a measure of how acidic the interior of the cell is—during each cell cycle. These pH oscillations coordinate the release of amino acids just when they are needed to supply the daughter cell with nutrients. Otherwise uncontrolled release of amino acids could poison the mitochondria, leading to defects in metabolism and other problems linked to disease and aging. Thus, the pH oscillations—and their breakdown as cells age—may be key to understanding a whole host of diseases of aging, the researchers say.
Everybody had previously thought of vacuoles as a terminal compartment where things are degraded, explains Patricia Kane, a biochemist at SUNY Upstate Medical University, who studies vacuolar ATPase, the enzyme that pumps protons into the vacuole. Now we see that vacuoles are not just a terminal compartment for waste processing but also a store of nutrients that the cells draw on when in need, such as during cell growth and division.
And “pH is something very intriguing, and it’s something that was neglected for a long, long time,” adds Joris Winderickx of KU Leuven in Belgium, who studies nutrient sensing and signaling in yeast.“The difficulty there was ‘How can you measure pH in an organelle?’ Now we have the tools to do that,” he explains, referencing the Calico Labs team’s development of a molecule whose fluorescence level corresponds to the acidity of the yeast vacuole. This allowed the researchers to detect and track changes in pH in the vacuole in real time.
About a decade ago Dan Gottschling, and Adam Hughes, both then at the Fred Hutchinson Cancer Research Center, discovered that aging yeast cells’ vacuoles slowly lose the ability to maintain their low pH. Hughes and Gottschling observed that this loss of vacuolar acidity causes mitochondrial dysfunction and aging (yeast cells typically have a life span of about 30 cell divisions). Preventing the loss in vacuolar acidity reduced mitochondrial dysfunction and extended the life span of the yeast cells.
The researchers found that the loss of vacuolar acidity means the amino acid transporters on the vacuole membrane can’t do their job. This leads to an uncontrolled release of amino acids in the cytosol, which poisons the mitochondria. Too much of the amino acid cysteine “botches up” the formation of iron-sulfur clusters that are essential to mitochondria. (Mitochondria need iron to make the energy-carrying molecule adenosine triphosphate, or ATP.) Without the iron-sulfur clusters, mitochondrial function becomes severely compromised: the mitochondria begin to produce large amounts of reactive oxygen species, which leads to aging and defects in DNA repair.
It’s clear that cells are not just using vacuoles as a storage depot, says Adam Hughes, who now leads a team at the University of Utah. The vacuoles do not only supply amino acids when they are needed. Just as important, the vacuoles sequester amino acids when they aren’t needed to prevent them from wreaking havoc.
Gottschling, now a distinguished principal investigator at Calico Labs, leads a team that has now discovered that the vacuoles in yeast cells not only lose their acidity over time as cells age but also show amazingly dynamic pH oscillations with every cell division. The fluctuating pH coordinates amino acid release with the varying metabolic needs at different phases of the cell cycle. The researchers developed a sensitive chemical sensor to visualize these oscillations inside vacuoles during cell division for the first time.
The researchers observed that the vacuole pH is low (very acidic) when a new cell bud first emerges and then rises—it becomes more alkaline—right before the daughter cell separates from the mother cell. In this way, the vacuole can control exactly when amino acids are released throughout the cell cycle and can provide nutrients to the daughter cell immediately before it buds off.
“A dividing cell needs those nutrients in bursts during cell division,” Gottschling explains. But when it is not dividing, the cell needs to keep amino acids safely sequestered to avoid damaging the mitochondria.
To probe how the pH oscillations were controlled, the researchers tried different ways to manipulate them. For example, the team found that when yeast cells were grown in the absence of amino acids, the pH oscillations were eliminated. They could be restored when the researchers added amino acids back to the growth medium. Similarly, a drug that inhibited TOR (target of rapamycin) signaling—one of the main nutrient-sensing cellular pathways—interrupted the pH oscillations. The researchers further found that in yeast mutants that lacked pH oscillations, the cells upregulated enzymes that were involved in amino acid biosynthesis. That suggests that the cells sensed amino acid starvation. But given that they could not release any of the sequestered amino acids from the vacuole, their only alternative was to make amino acids from scratch instead.
Although these pH oscillations are an exciting mechanism by which cells coordinate nutritional availability with the cell cycle, many open questions remain. For instance, it isn’t clear exactly how these pH oscillations are regulated, nor is it completely known why cells lose the ability to control pH oscillations in the vacuole as they age. Figuring out how this process breaks down could provide clues about how to intervene and prevent the cascading negative impacts on mitochondrial function and DNA repair.
“We see that [process] breaking down, and one of the things we want to figure out is ‘How does it break down?’” Gottschling says. “That’s why we are excited about what’s being sensed [to cause these oscillations] and then ‘How does that sensing break down?’”
Even though questions remain, there’s no doubt that these oscillations—and their loss with age—are key to understanding a variety of diseases of aging, says Winderickx, who uses yeast as a model to study Alzheimer’s disease, Parkinson’s disease and cancer. There are a variety of mechanisms by which loss of vacuolar acidity can cause an illness. First, the loss of acidity prevents the cell from clearing out old or defective proteins, and their accumulation can lead to the protein plaques that are characteristic of Alzheimer’s. In addition, the loss of acidity and the concomitant release of amino acids that poison the mitochondria cause defects in mitochondrial metabolism. The production of too many reactive oxygen species can damage DNA and cause mutations that snowball into more cellular defects.
Winderickx suspects that these pH oscillations are probably not restricted to the vacuole because the organelle interacts closely with mitochondria and the network of membranes called the endoplasmic reticulum. Through this process, the different types of organelles communicate and exchange nutrients. It would be surprising if the pH level in other cellular organelles isn’t synchronized with the pH in the vacuoles. This would have a wide-ranging impact on cell function. Cellular enzymes each have a narrow range of pH in which they function optimally, so pH oscillations will cause enzyme activity in different cellular compartments to shift, sometimes dramatically. “A lot of diseases originate from an organelle which is malfunctioning, but this malfunctioning might be pH-related in many cases,” Winderickx says.