V-ATPases are proton pumps that hydrolyse ATP to transfer protons across a cell membrane. The proton pumps drive a whole host of biological transport processes, including nutritional absorption across the Manduca Sextalarva midgut. When Manduca larvae moult, they shed the old gut lining and starve until the gut tissue has regenerated, so they don't need to fuel nutritional absorption until moulting is complete. When Hans Merzendorfer joined Helmut Wieczorek's group in Osnabrück, he wondered whether the hormones that control moulting would also regulate the amount of V-ATPase that the insect's gut makes. Together with Stephan Reineke, Merzendorfer found that the moulting hormone, ecdysterone, switches off V-ATPase synthesis, just when the insect needs the protein least (p. 1059).
Manduca larvae have high levels of V-ATPase because they have voracious appetites, and use 10% of their energy to drive nutritional absorption. One way of conserving energy while the insect moults is to shut down the midgut V-ATPase proton pump. But it is even more effective to stop V-ATPase synthesis all together. Could ecdysteroid hormones, which trigger moulting, also regulate V-ATPase synthesis?
Reineke and Merzendorfer began testing whether V-ATPase mRNA levels were affected when the insects were starved or moulting. When Reineke found that mRNA levels dropped in response to both situations, he gave larvae doses of two hormones that control moulting: ecdysterone and juvenile hormone. The mRNA levels for 11 of the 12 V-ATPase protein subunits dropped in response to ecdysterone. But knowing that cellular levels of mRNA fall in response to moulting hormones doesn't tell you how the hormones regulate the drop. Merzendorfer realised that he needed to clone some of the V-ATPase genes before he could find out how the hormone controlled the transporter.
Merzendorfer had to build a genomic library before he could clone three of the 12 subunit genes. Six months later, he had his clones, and the DNA evidence that all three genes carried the key DNA sequence that ecdysteroids interact with to control transcription of all three genes.
Once Merzendorfer knew that the genes had the correct ecdysteroid switch,he had to rule coincidence out by proving that the ecdysteroids were switching off mRNA synthesis. He attached the DNA switch from a VATPase subunit gene to a light emitting protein gene, and put the hybrid gene into experimental cells where the DNA switch allowed the cells to make the glowing protein. But when he exposed the cells to ecdysterone, the light went out. The hormone was controlling the levels of the light emitting protein in exactly the same way as it controlled V-ATPase genes in the moulting larva.
Ecdysterone was already known to regulate many essential proteins during the insect's metamorphosis. Now that list includes V-ATPase genes, thanks to Wieczorek's team.