Studies of Cytochrome c Oxidase-Driven H⁺-Coupled Phosphate Transport Catalyzed by the Saccharomyces cerevisiae Pho84 Permease in Coreconstituted Vesicles

Abstract

Studies of cytochrome c oxidase-driven H+-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles Fristedt, U.; van der Rest, M.E.; Poolman, Berend; Konings, W.N; Persson, B.L. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1999Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Fristedt, U., van der Rest, M. E., Poolman, B., Konings, W. N., & Persson, B. L. (1999 6, 1999; ReVised Manuscript ReceiVed September 16, 1999 ABSTRACT: The proton-coupled Pho84 phosphate permease of Saccharomyces cereVisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.The H + /phosphate cotransporter (Pho84p) of Saccharomyces cereVisiae is a polytopic membrane-spanning protein of the plasma membrane that catalyzes the coupled translocation of P i and H + according to a symport mechanism (see refs 1-3 for reviews). This high-affinity transporter is encoded by the PHO84 gene, which has been cloned and sequenced (4), shown to be derepressible by phosphate starvation, and regulated at the transcriptional level by the PHO regulatory pathway (5, 6). Its synthesis is repressed by P i concentrations of >100 µM (6). On the basis of hydropathy profile analysis of the primary amino acid sequence, a secondary structure model is proposed in which 12 transmembrane domains traverse the membrane in a zigzag fashion connected by hydrophilic loops, and with both N-and C-termini on the cytoplasmic side of the membrane (2). As in the case of several HXT sugar transporters (see refs 7 and 8 for recent reviews), the Pho84p exhibits a significant number of conserved residues between each of the two halves of the transporter, separated by the large central loop harboring 75 amino acids, suggesting that the protein has arisen from a duplication of an ancestral gene (2, 3). The protein has been proposed to belong to the phosphate:H + symporter (PHS) family of functionally uniform members (9). Construction of a His-tagged version of the Pho84p has allowed for stable expression of the protein in the cytoplasmic membrane of Escherichia coli. The protein, solubilized from the membrane, purified, and reconstituted into liposomes, was shown to catalyze an uncoupler-s...

The Saccharomyces cerevisiae High Affinity Phosphate Transporter Encoded by PHO84 Also Functions in Manganese Homeostasis

“…Recombinant Pho84p was found to require the presence of metal ions such as manganese and cobalt for phosphate transport, and a MeHPO 4 metal-phosphate complex was the proposed substrate. Not all metal phosphate complexes are good substrates for Pho84p, as the addition of magnesium was shown to actually have an inhibitory effect on phosphate transport with recombinant Pho84p (47). Consistent with this, we found an inhibitory effect of magnesium on the Pho84p-dependent uptake of manganese in vivo (data not shown).…”

“…Evidence for such a transport mechanism has come from studies of recombinant Pho84p in reconstituted proteoliposomes (47). Recombinant Pho84p was found to require the presence of metal ions such as manganese and cobalt for phosphate transport, and a MeHPO 4 metal-phosphate complex was the proposed substrate.…”

“…These bacterial phosphate transporters show a striking biochemical similarity to Pho84p. The substrate for both Pho84p and the bacterial phosphate transporters are thought to be neutral metal-phosphate complex, and each of these transporters shows a preference for MnHPO 4 (47)(48)(49).…”

Regulation of Cation Balance inSaccharomyces cerevisiae

“…The transport substrate of Pho84 is a complex of phosphate anion and divalent metal cation (Fristedt et al 1999). In standard laboratory media, magnesium concentrations are relatively high, and the substrate for Pho84 is likely a MgHPO 4 complex.…”

Manganese Transport and Trafficking: Lessons Learned from Saccharomyces cerevisiae

“…Using reconstituted proteoliposomes, Fristedt and colleagues have shown that recombinant Pho84p can transport metal-phosphate (MeHPO 4 ) complexes, with manganesephosphate being a particularly good substrate (21). It is therefore conceivable that when yeast cells are exposed to very high levels of manganese, the manganese-phosphate complexes that form in the environment are readily taken up via the Pho84p transporter (Fig.…”