Adaptation to fungal cold linked to changes in protein structure: study
Jthere is only a certain amount of stress that biological structures can withstand before collapsing. Tissue fluids freeze into ice crystals when exposed to temperatures below -3°C, for example, and enzymes break down and become dysfunctional at extremely low temperatures. But some organisms are designed to thrive in such harsh conditions. Some species of fungi, for example, can survive the harsh Antarctic weather, and scientists have spent years trying to figure out how. A study published in Scientists progress on September 7 suggests that these polar organisms may have adapted due to changes in the unstructured regions of their proteins.
Intrinsically disordered regions (IDRs) of proteins are the shapeless, liquid parts of proteins that lack the ability to fold into a functional shape and often react with RNA to form naked or membraneless cellular organelles such as the nucleolus by a phenomenon known as liquid-liquid phase separation (LLPS).
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The study shows that yeasts adapted to severe cold experienced evolutionary changes in IDRs, which alter the way phase separation occurs. The researchers found that the structure of IDRs in species adapted to polar regions is different from those in temperate regions.
The researchers stumbled upon these differences while studying how transcription occurs in the cold. They analyzed two main components of the transcription system of the RNA polymerase II multisubunit enzyme – the carboxy-terminal domain (an IDR) and the prolyl isomerase Ess1 – in five isolated cold- and salt-adapted yeast species. from the Arctic and Antarctic, when they noticed that the carboxy-terminal domain (CTD) structure of the species was slightly different from that of the model yeast species baker’s yeast (Saccharomyces cerevisiae).
Steve Clabuesch, a former colleague of study co-author Steven Hanes, walks along Commonwealth Glacier, McMurdo Dry Valleys, Antarctica in 2016.
In baker’s yeast, the CTD consists of a repeating peptide sequence – YSPTSPS – whereas the repeating sequences in polar yeasts diverge at positions one, four and seven. Study co-author Steven Hanes, a molecular geneticist at the SUNY Upstate Medical University in New York says the repeating sequence is shared by vast clades of life and is nearly identical even in humans, so this divergence in cold-adapted yeasts was “extremely significant.” “He and his colleagues were curious as to why CTDs from polar yeasts showed such differences and wondered if these CTDs would still work in baker’s yeast.
First, Hanes and colleagues deleted the gene that encodes the CTD from baker’s yeast but kept the CTD intact by retaining the plasmid that expresses the enzyme Rpb1 (a subunit of RNA polymerase II), which has kept the host cell alive. Next, they cloned a polar yeast CTD gene into a plasmid and transferred it to baker’s yeast. They did this to test whether the divergent CTD would work when paired with the structured region of RNA polymerase II in baker’s yeast. The procedure was performed at 18°C and 30°C to determine the effects of colder temperatures.
Hanes explains that if the cloned CTD gene is compatible with the host, it will evict the original plasmid from the host cell in favor of the new one. The degree to which the originals are lost will give an estimate of the degree of compatibility of the cloned plasmid with the model species.
Baker’s yeast replaced its own plasmid fairly well with that of the various polar yeasts at 30°C. But at 18°C, baker’s yeast containing CTDs from arctic mushrooms Wallemia ichthyophaga, Aureobasidium pullulansand Hortaea werneckii lost only 0.2%, 13.6%, and 21.5%, respectively, while those containing CTDs from Antarctic fungi Cryoxeric dioszegia and Naganishia vishniacii did not lose any of the original plasmids. In contrast, the control lost 58% and 87% of the original plasmid at 18°C and 30°C, respectively. The researchers report that the polar yeast CTD did not work in baker’s yeast at 18°C.
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Hanes says he suspects the differences may stem from the mechanisms of LLPS, as a previous study found that CTDs can undergo the process. The team therefore checked whether the CTDs of cold-adapted yeast could also undergo this phenomenon.
The researchers linked polar CTDs with purified proteins in a test tube and checked for the presence of LLPS by observing the turbidity of the solution – proof that LLPS occurred – at different temperatures and salinity levels. Hanes and his team observed that the CTDs of these polar yeasts undergo phase separation, but they do so differently from baker’s yeast. They noticed that the CTDs of the species most compatible with S.cerevisiae at 18°C showed elevated LLPS, while those that were not compatible showed none. The researchers attributed these different properties to the discrepancy in the amino acid sequence of the CTDs, and they hypothesize that this discrepancy may promote cold and salt tolerance in polar species.
Hanes says that intrinsically disordered regions of the protein with more variable sequences are highly adaptive to “selective pressures that would alter the biophysical properties of how proteins sort in cells.” And this adaptation can change how, when and where they undergo phase separation.
“We know that stress can induce phase separation by certain proteins, but what we suggest is that environmental tuning of phase separation allows organisms to tolerate temperature and other extreme conditions,” explains Hanes.
Amy Gladfelter, a cell biologist at the University of North Carolina who studies phase separation and did not work on the new study, says the results “really suggest that by looking at natural variation and . . . at the how free yeasts can adapt to extreme temperatures and also to extreme salinity, [the research team] can find evidence of adaptation in sequences that are important in leading to phase separation.
Hanes tells The scientist that the study uncovered more questions than answers, but the team intends to answer most of them, including the exact mechanism by which phase separation properties confer environmental tolerance, as this could help other microorganisms to survive in harsh and changing climatic conditions.