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Abstract

One outcome of compartmentalization in eukaryotic cells is that each organelle has the ability to establish a unique pH environment. Homeostasis of the luminal pH within these organelles is essential for survival of the cell because important processes such as secondary active transport and enzymatic reactions depend on optimal pH conditions. Luminal pH is a product of the cooperative effort between H+ pumps and ion transporters located on the limiting membrane of the organelle. The vacuolar H+-ATPases (V-ATPases) are one class of proton pumps that are well conserved within eukaryotic cells. V-ATPases are multisubunit complexes that hydrolyze ATP and transport protons into the lumen of organelles. In plant cells, the V-ATPase is important for pH homeostasis at the vacuole and early endosome but most of the information available about the eukaryotic V-ATPase complex comes from studies in yeast and mammals. In plants, nothing is known about the mechanism of targeting in the cell, the regulation of its activity and whether the pump has secondary functions aside from pH homeostasis. Our research focus was on subunit a (VHA-a) of the VO subcomplex of plants. The localization of the V-ATPase in the cell is determined by the isoforms of VHA-a. The sorting signal of any VHA-a subunit was not known. In the first chapter, we report the discovery of an acidic cluster and a critical leucine (L159) residue in the N-terminus of VHA-a1 that serves as both an ER export signal and as a TGN retention motif. This motif is reminiscent of mammalian endosomal targeting acidic clusters but the specific sequence of amino acids is unique to the plant kingdom and the motif originates in the gymnosperm sequences. In the second chapter we investigated whether the TGN localized V-ATPase not only fulfils its primary function of acidification, but whether it is also a component of the molecular machinery that senses the level of luminal acidification and its recruits cytosolic proteins involved in vesicle trafficking to the membrane. In the third chapter, we explore a new form of modification of the tonoplast localized isoform (VHA-a3). We show that Sacylation is not involved in targeting of VHA-a3 to the tonoplast and that VATPase activity is not compromised by a lack of S-acylation in the presence of high concentrations of zinc.