Genetic Manipulation of Vacuolar Proton Pumps and Transporters,

Abstract

Cells expend as much as 50% of their total intra- cellular energy reserves to maintain gradients of ions across their membranes (Nelson, 1994). These gradi- ents have been associated with the myriad of func- tions attributed to the membranes of living organ- isms. In the past, much of our knowledge about the function of the proteins involved in creating these ion gradients came from biochemical and biophysical studies. However, it was often difficult to associate the vast repertoire of membrane functions with par- ticular proteins. Now, with the complete sequence of the Arabidopsis and yeast (Saccharomyces cerevisiae) genomes, and the facility with which genes can be engineered and transferred between these two organ- isms, there are new opportunities to identify each transporter encoded in the genome with a specific set of functions in the organisms. These new tools have also made it possible to examine the basic tenets of the chemiosmotic hypothesis in intact organisms. Plants and fungi are similar in that they use pro- tons as the "currency" (proton electrochemical gra- dient (PEG)) with which to mediate ion gradients (Sze et al., 1999), whereas animal cells use Na ions as the driving force. While plants work at photosyn- thesis, they are making an important long-term in- vestment by creating H gradients. The initial "cash reserve" is generated by transport systems that form the H gradient across biological membranes. Be- cause these pumps invest the plant's energy, they are likely to be tightly controlled (Sagermann et al., 2001). The accumulation of ions into intracellular organelles (vacuoles, prevacuolar bodies, and Golgi vesicles) against the concentration gradient often re- quires the "withdrawal" of the H currency from an intracellular organelle by the secondary transporters. Each of the secondary transporters (e.g. proton/cat- ion antiporters) can be thought of as an individual company that imports and exports goods and ser- vices. Thus, a hierarchy is formed between the H pumps that inject the currency into the intracellular organelles and the secondary transporters that utilize this currency. In this survey, we will consider only the role of the two vacuolar H pumps in the gener- ation of the PEG, although we are aware that the magnitude of the PEG can be affected by other trans- porters (i.e. H cotransporters and electrogenic anti-