Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses a huge amount of energy in form of ATP, to pump out protons. To avoid complete energy depletion in the cells, tight regulation of the PM H+-ATPase is a necessity. The proteins two terminal domains have been identified as autoinhibitory domains that regulate the pumping activity, but due to lack of a high resolution 3D structure the mechanism behind is only poorly understood. This thesis aimed at illuminating the autoinhibitory mechanism in plant and yeast PM H+-ATPases and below some of our main findings will be highlighted.

The two terminal domains of the PM H+-ATPases have several amino acid residues that can be phosphorylated, and it has been demonstrated that these phosphorylation sites in both plant and yeast are highly involved in the regulation of terminal autoinhibition. In this study we used a phylogenetic analysis to investigate the evolutionary development of these phosphorylation sites. In contrast to fungal PM H+-ATPases the terminal phosphorylation sites in the plant counterpart was found highly conserved even in the earliest land plants. The phosphorylation sites were, however, not found in algae, the sister group of land plants. We therefore hypotheses that the delicate regulation of plant PM H+-ATPases developed with the first land plants and has remained conserved ever since. Beside phosphorylation in the terminal domains, lipid homeostasis also influences the autoinhibitory regulation. A group of lipids called lyso-phospholipids have been identified as signaling molecules in mammalian cells and it has been speculated if they have a similar function in plants. In this thesis we show, that plant PM H+-ATPases are receptors for lysophospholipids and the autoinhibitory terminal inhibition is released upon lysophospholipid binding. Finally, we have used a group of stabilizing compounds called metal fluoride, to lock the yeast PM H+-ATPase in different states in the catalytic pumping cycle, and studied how the autoinhibitory terminal domains change the overall protein conformation.