Study explores mitochondrial-nuclear communication pathway

In an article published by the journal Free Radical Biology and Medicine, researchers show what happens in a yeast cell when one of the signaling pathways between mitochondria and nuclei is impaired.

By Karina Toledo

Agência FAPESP – Considered highly important organelles in eukaryotic cells because they act as cell “powerhouses,” mitochondria must be in constant communication with cell nuclei, where the proteins that they require are encoded.

Communication follows several different pathways. In the most studied of these pathways, the signal leaves the nucleus and travels to the mitochondrion. In other, less well-known pathways, the signal moves in the opposite direction. For this reason, these pathways are known as retrograde signaling pathways.

In experiments with yeast of the species Saccharomyces cerevisiae, which is the type used to make bread, beer, wine and cheese, Brazilian researchers have investigated for the first time what happens to mitochondria when one of these retrograde pathways, mediated by Rtg proteins, does not work properly.

The research was conducted at the Center for Research on Redox Processes in Biomedicine (Redoxome), one of FAPESP’s Research, Innovation and Dissemination Centers (RIDCs). The findings were recently published in the journal Free Radical Biology and Medicine.

“We observed various differences in cells without an active retrograde signaling pathway. These differences included higher oxygen consumption by mitochondria and greater cellular susceptibility to oxidative stress,” said Fernanda Marques da Cunha, a professor at the Federal University of São Paulo (UNIFESP) and the principal investigator of the project supported by FAPESP. Oxidative stress is a condition characterized by an increase in the levels of reactive oxygen species, which can damage molecules that are important for cell balance.

According to Cunha, several proteins act as mediators of retrograde signaling pathways. In the case of the Rtg-mediated pathway, previous research by other groups has identified three proteins as the most important: Rtg1, Rtg2, and Rtg3.

“All three are found in the cytoplasm. Rtg2 activates Rtg1, which binds with Rtg3 to form a transcription factor that moves to the nucleus and activates several genes involved in the mitochondrial metabolism,” Cunha said.

To find out what would happen to the mitochondria if this communication were impaired for some reason, the researchers compared cultured wild-type yeast cells in which all three proteins functioned normally with two types of mutant yeast, one in which the gene encoding Rtg1 was silenced, and another in which the gene for Rtg2 was missing.

The cells were cultured in a glucose-rich medium for seven days and evaluated after this period.

“When we analyzed the cells, we found that the two mutant strains consumed approximately twice as much oxygen as the intact wild-type yeast. This was unexpected. We thought that the mitochondrial metabolism would be damaged and that less oxygen would be consumed,” Cunha said.

Another experiment was performed to assess whether this high oxygen consumption was linked to a larger amount of mitochondria in the cells.

“During these seven days, the cells pass through different phases. Initially, they obtain energy anaerobically by fermenting glucose. When this phase ends, they consume fermentation products aerobically, and the number of mitochondria inside the cells increases. When all of the respiratory substrates are depleted, the cells enter a stationary phase. They stop dividing and lower their respiration levels, decreasing the number of mitochondria,” Cunha said.

The researchers at Redoxome found that although the mutant yeast cells also entered a stationary phase after the seventh day of culture, the number of mitochondria inside these cells did not decrease as it did in the wild-type yeast cells. Moreover, in proportional terms, each organelle consumed far more oxygen than its counterparts in the wild-type cells.

“When the cells’ demand for mitochondria decreases, these organelles are selectively degraded in a process known as mitophagy. We showed that mitophagy was reduced in the mutant cells,” Cunha said.

What doesn’t kill you makes you stronger

When the mitochondria are not working well, she explained, they produce more hydrogen peroxide (H2O2), which is one of the causes of oxidative stress.

However, when the researchers isolated the mitochondria in the mutant cells, they found that H2O2 production was lower than in the wild-type cells.

“There is a concept known as hormesis, according to which very small amounts of toxic substances are beneficial to the organism because they stimulate defense mechanisms that help it prepare to address larger doses. As the saying goes, what doesn’t kill you makes you stronger,” Cunha said.

To test whether this concept applied to the yeast cells being studied, the researchers challenged the cells by placing them in a medium with high H2O2 concentrations. Confirming their hypothesis, the wild-type cells survived approximately three times longer than the mutant cells.

“The mutant cells displayed a higher capacity to convert H2O2 into non-toxic substances such as oxygen and water. In both cell types, there was less activity of glutathione peroxidase, one of the enzymes responsible for neutralizing H2O2. There was also less catalase enzyme activity in the cells without the Rtg1 protein,” Cunha said.

The experiments were performed during Nicole Quesada Torelli’s research for her master’s degree at the University of São Paulo’s Chemistry Institute (IQ-USP), supervised by Cunha and Alicia Kowaltowski, another professor at USP. José Ribamar Ferreira Júnior, a researcher affiliated with USP’s School of Arts, Sciences & Humanities (EACH), also collaborated.