Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display.

Retroviral particles are expensive to manufacture, mostly due to the downstream processing steps which result in low recoveries (≈30%) and concentration factors. In this work, a dodecapeptide phage-display library was panned against retrovirus like particles expressing the envelope protein Ampho4070A (VLPs-AMPHO) and VLPs without the target protein, used as a negative control (VLPs). A depletion/selection panning protocol was successfully used to deal with the structural complexity of the target, and a total of three distinct peptide sequences displaying preferential binding towards VLPs-AMPHO were found. Peptide 3 (CAAALAKPHTENHLLT), which appeared as one lead candidate, was synthesized and immobilized onto two purification matrices, cross-linked agarose and magnetic particles. The matrices selectively bound VLPs-AMPHO and in both cases recovery yields higher than 90% were obtained when employing mild elution conditions, while maintaining viral particle morphology and size.

Inorganic Phosphate and Sulfate Transport in S. cerevisiae.

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.

An externally accessible linker region in the sodium-coupled phosphate transporter PiT-1 (SLC20A1) is important for transport function.

BACKGROUND/AIMS: Members of the SLC20 cotransporter family (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform housekeeping functions for intracellular Pi homeostasis as well as being implicated in vascular calcification and renal Pi reabsorption. The aims of this study were to investigate the topology of a linker region in PiT-1 between the predicted 2(nd) and 3(rd) transmembrane domains and to investigate the functional consequences of cysteine substitutions in this region. METHODS: Cysteines were substituted at 18 sites in the Xenopus PiT-1 isoform and the mutants were expressed in Xenopus laevis oocytes. Transport function of the mutants was investigated by (32)P tracer or two electrode voltage clamp before and after thiol modification of the novel Cys. RESULTS: Exposure to the thiol reactive reagent resulted in diminished transport function for 7 mutants. The apparent accessibility of 5 of the mutated sites, estimated from the rate of functional thiol modification, was site-dependent. Cysteine substitution at some sites also altered the apparent affinity for Pi and cation (Na(+)/Li(+)) and substrate (phosphate/arsenate) selectivity, further underscoring the importance of this linker in defining PiT-1 transport characteristics. CONCLUSIONS: The external accessibility of a linker in PiT-1 was confirmed and sites were identified that determine substrate selectivity and transport function.

Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na(+)/Pi 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.

Functional expression, purification and reconstitution of the recombinant phosphate transporter Pho89 of Saccharomyces cerevisiae.

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, respectively. 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.

Phosphate transport kinetics and structure-function relationships of SLC34 and SLC20 proteins.

Transport of inorganic phosphate (P(i)) is mediated by proteins belonging to two solute carrier families (SLC20 and SLC34). Members of both families transport P(i) using the electrochemical gradient for Na(+). The role of the SLC34 members as essential players in mammalian P(i) homeostasis is well established, whereas that of SLC20 proteins is less well defined. The SLC34 family comprises the following three isoforms that preferentially cotransport divalent P(i) and are expressed in epithelial tissue: the renal NaPi-IIa and NaPi-IIc are responsible for reabsorbing P(i) in the proximal tubule, whereas NaPi-IIb is more ubiquitously expressed, including the small intestine, where it mediates dietary P(i) absorption. The SLC20 family comprises two members (PiT-1, PiT-2) that preferentially cotransport monovalent P(i) and are expressed in epithelial as well as nonepithelial tissue. The transport kinetics of members of both families have been characterized in detail using heterologous expression in Xenopus oocytes. For the electrogenic NaPi-IIa/b, and PiT-1,-2, conventional electrophysiological techniques together with radiotracer methods have been applied, as well as time-resolved fluorometric measurements that allow new insights into local conformational changes of the protein during the cotransport cycle. For the electroneutral NaPi-IIc, conventional tracer uptake and fluorometry have been used to elucidate its transport properties. The 3-D structures of these proteins remain unresolved and structure-function studies have so far concentrated on defining the topology and identifying sites of functional importance.

Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life.

BACKGROUND: The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na(+)- or H(+)-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na(+)-dependent P(i) (NaP(i)) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. RESULTS: We show that the human PiT2 histidine, H(502), and the human PiT1 glutamate, E(70),--both conserved in eukaryotic PiT family members--are critical for P(i) transport function. Noticeably, human PiT2 H(502) is located in the C-terminal PiT family signature sequence, and human PiT1 E(70) is located in ProDom domains characteristic for all PiT family members.A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR(254)-V(483)), was found to be a fully functional P(i) transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL(183)-V(483)) did also support P(i) transport albeit at very low levels. CONCLUSIONS: The results suggest that the overall structure of the P(i)-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.

New structural arrangement of the extracellular regions of the phosphate transporter SLC20A1, the receptor for gibbon ape leukemia virus.

Infection of a host cell by a retrovirus requires an initial interaction with a cellular receptor. For numerous gammaretroviruses, such as the gibbon ape leukemia virus, woolly monkey virus, feline leukemia virus subgroup B, feline leukemia virus subgroup T, and 10A1 murine leukemia virus, this receptor is the human type III sodium-dependent inorganic phosphate transporter, SLC20A1, formerly known as PiT1. Understanding the critical receptor functionalities and interactions with the virus that lead to successful infection requires that we first know the surface structure of the cellular receptor. Previous molecular modeling from the protein sequence, and limited empirical data, predicted a protein with 10 transmembrane helices. Here we undertake the biochemical approach of substituted cysteine accessibility mutagenesis to resolve the topology of this receptor in live cells. We discover that there are segments of the protein that are unexpectedly exposed to the outside milieu. By using information determined by substituted cysteine accessibility mutagenesis to set constraints in HMMTOP, a hidden Markov model-based transmembrane topology prediction method, we now propose a comprehensive topological model for SLC20A1, a transmembrane protein with 12 transmembrane helices and 7 extracellular regions, that varies from previous models and should permit approaches that define both virus interaction and transport function.