Inorganic phosphate (Pi) uptake is essential to all cells meeting metabolic requirements for growth and replication. Transport of phosphate across the cell plasma membrane is the first, obligatory step of phosphate utilization in the cell. In the yeast Saccharomyces cerevisiae this uptake is regulated by the PHO system, i.e., a network of scattered genes involving structural and regulatory units [17, 16]. This complex is composed of at least five gene products through which signals in the presence or absence of a repressible amount of phosphate are conveyed, and operates through two major modes by which phosphate uptake across the plasma membrane is mediated. Of these, one low-affinity system is active at high concentrations and one high-affinity system is activated at low concentrations of extracellular phosphate [14]. These systems are permeases, which are secondary co-transporters driven by a transmembrane ion gradient, e.g., a concentration gradient and/or an electrical gradient [9]. The low-affinity system, with a Km for phosphate of approximately 1 mM at its proposed optimum of pH 4.5, is considered to be a constitutively expressed Pi/H+ co-transporter essential for survival of the yeast cell [11, 17]. In contrast, the high affinity system is derepressible by phosphate starvation during aerobic and anaerobic cell growth. Of the proteins responsible for the high-affinity transport of phosphate into the cell, one is Pho89, a Na+-coupled phosphate transporter with a pH optimum of 9.5 [8]. Pho89 is a 63-kDa protein composed of 574 amino acids organized as 12 putative transmembrane segments that traverse the membrane in a zigzag fashion, connected by more hydrophilic domains. The other is Pho84, a Pi/H+ co-transporter with a pH optimum for phosphate uptake similar to that of the constitutive low-affinity system [1, 2].

Pho84 is a 65kDa protein composed of 587 amino acids and contains like the Pho89 protein 12 putative transmembrane segments, traversing the membrane, but with more extended hydrophilic N- and C-termini than the Pho89 protein. The activity of the Pho84 protein has been proposed to rely on a number of additional gene products, i.e. Pho86, Pho87, Pho88, and Gtr1, a putative GTP-binding protein [15]. These have been proposed to serve as a receptor for phosphate signals from the environment [12] or otherwise act as a regulatory unit in the adaptation of the transporters to changing metabolic conditions in the cell [15]. Although it is also possible that they are directly involved in the transport of phosphate across the plasma membrane in a complex with Pho84 it is clear that the Pho84 alone is capable of catalyzing the high-affinity transport of Pi across the plasma membrane [1, 4]. After synthesis, the Pho84 protein is transported to the plasma membrane [15] via the endoplasmatic reticulum (ER) and the Golgi compartments [7]. For this transport it has been shown that Pho86 is necessary for packaging of the Pho84 into COPII vesicles for transport between ER and the Golgi compartments. In this process, Pho86 is not itself present in the COPII vesicles, and is thus classified as an outfitter, a protein facilitating the embarkation of a secretory protein into vesicles for transport without being co-packaged itself [5].

The Gtr1 has been reported to have an intracellular function as well as taking part in the phosphate signaling from the environment and has been suggested to be a negative regulator of the Ran/Gsp1 GTPase cycle [10]. Recent studies suggest that the activities of the Pho84 and Pho89 proteins are individually regulated [13]. The activation and inactivation of the Pho89 protein transport activity occurs early in the growth phase with a maximum activity at OD600 of 0.5 following induction of the PHO89 gene transcript at external Pi concentrations as high as 350 mM. The PHO89 gene is maintained induced also when the Pho89 transport activity is close to completely inactivated at higher OD600 values in the range 1.5 to 3. This indicates that the Pho89 transport activity is regulated by the amount of available inorganic phosphate and not strictly by expression [13]. This in contrast to the activity of the Pho84, where induction of the protein in response to Pi starvation correlates with the increase in Pi uptake rate, and thus appear to be controlled at transcriptional level. In contrast to the Pho89 protein, the Pho84 protein has its activity maximum of transport at OD600 of 2. The induction of the PHO84 transcript, and synthesis of the transporter, requires a external Pi concentration below 100 mM [2]. When the functional Pho84 protein meets a limitation in external Pi concentration, the protein disappears from the plasma membrane to be transferred to the vacuole where the protein undergoes degradation. This may possibly reflect a cellular strategy to prevent efflux of essential internalized phosphate [6]. Interestingly, the phenomenon is also seen when repressible amounts of phosphate are added to phosphate-starved cells, in which a rapid removal of the Pho84 from the plasma membrane followed by vacuolar localization occurs [7]. Whether this possibly reflects a regulated strategy of the cells to prevent overloading of phosphate is not clear.

References
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