Inorganic ions such as phosphate and sulfate are essential macronutrients required for a broad spectrum of cellular functions and their regulation. In a constantly fluctuating environment microorganisms have for their survival developed specific nutrient sensing and transport systems ensuring that the cellular nutrient needs are met. This chapter focuses on the S. cerevisiae plasma membrane localized transporters, of which some are strongly induced under conditions of nutrient scarcity and facilitate the active uptake of inorganic phosphate and sulfate. Recent advances in studying the properties of the high-affinity phosphate and sulfate transporters by means of site-directed mutagenesis have provided further insight into the molecular mechanisms contributing to substrate selectivity and transporter functionality of this important class of membrane transporters.

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae. Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi: H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica. However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi. A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr(179) in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr(179), Asp(178), is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr(179) then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr(179) in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

Phosphorus (P) is a major macronutrient for plant health and development. The available form of P is generally low in the rhizosphere even in fertile soils. A major proportion of applied phosphate (Pi) fertilizers in the soil become fixed into insoluble, unavailable forms, which restricts crop production throughout the world. Roots possess two distinct modes of P uptake from the soil, direct and indirect uptake. The direct uptake of P is facilitated by the plant's own Pi transporters while indirect uptake occurs via mycorrhizal symbiosis, where the host plant obtains P primarily from the fungal partner, while the fungus benefits from plant-derived reduced carbon. So far, only one Pi transporter has been characterized from the mycorrhizal fungus Glomus versiforme. As arbuscular mycorrhizal fungi cannot be cultured axenically, their Pi transporter network is difficult to exploite for large scale sustainable agriculture. Alternatively, the root-colonizing endophytic fungus Piriformospora indica can grow axenically and provides strong growth-promoting activity during its symbiosis with a broad spectrum of plants. P indica contains a high affinity Pi transporter (PiPT) involved in improving Pi nutrition levels in the host plant under P limiting conditions. As P indica can be manipulated genetically, it opens new vistas to be used in P deficient fields.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of P_i across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na⁺/P_i cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate. 

The Saccharomyces cerevisiae high-affinity phosphate transporter Pho89 is a member of the inorganic phosphate (Pi) transporter (PiT) family, and shares significant homology with the type III Na⁺/Pi symporters, hPit1 and hPit2. Currently, detailed biochemical and biophysical analyses of Pho89 to better understand its transport mechanisms are limited, owing to the lack of purified Pho89 in an active form. In the present study, we expressed functional Pho89 in the cell membrane of Pichia pastoris, solubilized it in Triton X-100 and foscholine-12, and purified it by immobilized nickel affinity chromatography combined with size exclusion chromatography. The protein eluted as an oligomer on the gel filtration column, and SDS/PAGE followed by western blotting analysis revealed that the protein appeared as bands of approximately 63, 140 and 520 kDa, corresponding to the monomeric, dimeric and oligomeric masses of the protein, respec- tively. Proteoliposomes containing purified and reconstituted Pho89 showed Na⁺-dependent Pi transport activity driven by an artificially imposed electrochemical Na+ gradient. This implies that Pho89 operates as a symporter. Moreover, its activity is sensitive to the Na⁺ ionophore monensin. To our knowledge, this study represents the first report on the functional reconstitution of a Pi-coupled PiT family member. 

In the yeast Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events.

Autism Spectrum Disorders (ASD) are classified as neurological developmental disorders. Several studies have been carried out to find a candidate biomarker linked to development of these disorders, but up to date no reliable biomarker is available. Mass spectrometry techniques have been used for protein profiling of blood plasma of children with such disorders in order to identify proteins/peptides which may be used as biomarkers for detection of the disorders. Three differentially expressed peptides with mass charged (m/z) values of 2,020 ± 1, 1,864 ± 1, and 1,978 ± 1 Da in heparin plasma of children with ASD which were significantly changed as compared to the peptide pattern of the non-ASD control group are reported here. This novel set of biomarkers allows for a reliable blood based diagnostic tool that may be used in diagnosis and potentially, in prognosis of ASD. 

Autism spectrum disorders (ASDs) are neurodevelopmental and behavioural syndromes affecting social orientation, behaviour, and communication that can be classified as developmental disorders. ASD is also associated with immune system abnormality. Immune system abnormalities may be caused partly by complement system factor I deficiency. Complement factor I is a serine protease present in human plasma that is involved in the degradation of complement protein C3b, which is a major opsonin of the complement system. Deficiency in factor I activity is associated with an increased incidence of infections in humans. In this paper, we show that the mean level of factor I activity in the ASD group is significantly higher than in the control group of typically developed and healthy children, suggesting that high activity of complement factor I might have an impact on the development of ASD.

In Saccharomyces cerevisiae, the high-affinity phosphate transport system comprises the Pho84 and Pho89 permeases. The Pho89 permease catalyzes import of inorganic phosphate in a symport manner by utilizing Na+ ions as co-solute. We have addressed the functional importance of two glutamic acid residues at positions 55 and 491. Both residues are highly conserved amongst members of the inorganic phosphate transporter (PiT) family, which might be an indication of functional importance. Moreover, both residues have been shown to be of critical importance in the hPit2 transporter. We have created site-directed mutations of both E55 and E491 to lysine and glutamine. We observed that in all four cases there is a dramatic impact on the transport activity, and thus it seems that they indeed are of functional importance. Following these observations, we addressed the membrane topology of this protein by using several prediction programs. TOPCONS predicts a 7-5 transmembrane segment organization, which is the most concise topology as compared to the hPiT2 transporter. By understanding the functionality of these residues, we are able to correlate the Pho89 topology to that of the hPiT2, and can now further analyze residues which might play a role in the transport activity.