Membrane transport systems active in cellular inorganic phosphate (P(i)) acquisition play a key role in maintaining cellular P(i) homeostasis, independent of whether the cell is a unicellular microorganism or is contained in the tissue of a higher eukaryotic organism. Since unicellular eukaryotes such as yeast interact directly with the nutritious environment, regulation of P(i) transport is maintained solely by transduction of nutrient signals across the plasma membrane. The individual yeast cell thus recognizes nutrients that can act as both signals and sustenance. The present review provides an overview of P(i) acquisition via the plasma membrane P(i) transporters of Saccharomyces cerevisiae and the regulation of internal P(i) stores under the prevailing P(i) status.

TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.Chloroplasts perform oxygenic photosynthesis in algae and plants and have evolved by endosymbiosis from cyanobacteria. Chloroplasts have two distinct membrane systems, the double envelope surrounding the organelle and an internal membrane system named thylakoids. The envelope membrane represents the interface between the cytoplasm and chloroplast stroma, whereas the thylakoid membrane separates the stroma and the lumenal space. Altogether ϳ800 membrane proteins have been identified by proteomics in the envelope and thylakoid membranes of Arabidopsis thaliana (for reviews, see Refs. 1 and 2). As expected, the main function for the identified envelope proteins was transport of ions and metabolites, whereas photosynthesis was attributed to most of the identified thylakoid proteins. The major protein complexes in thylakoids are photosystems (PS) 4 I and II, the cytochrome b 6 f complex, and the proton-translocating ATP synthase. These photosynthetic complexes contain not only proteins but also pigments and other cofactors. Their assembly, activity, and removal require a large number of auxiliary, regulatory, and transport proteins (3, 4). Many biochemical reports pointed to the existence of transport activities in the thylakoid membrane, such as calcium transport (5), copper transport (6), anion channels (7), cation channels (8, 9), and nucleotide transport (10). Only the thylakoid copper transporter was identified at the genetic level in Arabidopsis (11). No hydrophobic proteins related to the above-mentioned transport activities were identified in the previous proteomic works on Arabidopsis thylakoid membranes (for a review, see Ref.2). Therefore, genetic strategies are required for identification and elucidation of their role in optimal function of the thylakoid.ATP is produced during the light-dependent photosynthetic reactions on the stromal side of the thylakoid membrane. Besides its utilization during CO 2 fixation in the stroma, ATP drives many energy-dependent processes in thylakoids, including protein phosphorylation, folding, import, and degradation.

Data Availability Individual level data for the human study can only be obtained via the Biobank of The Institute of Health and Welfare in Finland (https://thl.fi/en/web/thl-biobank). Next-generation sequencing data have been deposited in the SRA data base (PRJNA563975) and its processed counts data could be found in the Supplementary Dataset 1. The individual processed data from cell lines (Fig. 4 and 5), mice studies (Fig. 6) and human islet work (Fig. 7) are available in the Source Data files. Additional data supporting the findings of this study are available on request from the corresponding author.

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipiddependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other insolution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14 -98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.High density lipoprotein (HDL) 3 plays a central role in lipid metabolism as a principal facilitator of reverse cholesterol transport, a process wherein cholesterol is mobilized from peripheral tissues and delivered to the liver and steroidogenic organs. Low levels of HDL represent a major risk factor for cardiovascular disease, and there is growing interest in therapies that can enhance HDL plasma levels or its anti-atherogenic activity (1).Apolipoprotein A-I (apoA-I) comprises ϳ70% of the protein content of HDL and is a primary determinant of HDL structure, composition, and stability. ApoA-I is a prominent member of the exchangeable apolipoprotein class of proteins, and HDL derives a large portion of its functionality from the ability of apoA-I to sequester phospholipid and cholesterol and functionally interact with plasma enzymes and cellular receptors (for a recent review see Ref.2).In a previous study, we applied site-directed spin label electron paramagnetic resonance (SDSL-EPR) spectroscopy to determine the conformation of the C-terminal domain of apoA-I (3). This approach allowed us to both describe the structure of the lipid-free apoA-I C terminus (residues 163-241) and also report on the structural changes that arise because of lipidation. Using a similar approach, we have targeted the N-terminal one-third of apoA-I to identify regions of conformational flexibility and sites of inter-and intramolecular interaction. The unique genetic (4, 5), pathophysiological (6, 7), and structural (8, 9) features associated with the N terminus highlight the need to ascertain the structure and dynamics of this region of apoA-I in the presence and absence of lipid.The present SDSL-EPR analysis of residues 14 -98 of fulllength apoA-I reveals a mixed secondary structure composition for this region. We also identify sites of more stable tertiary interactions in an otherwise conformational dynamic segment of the protein. To observe the effect of lipid binding on the structure of the N t...

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free / lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-I IOWA ) leads to the formation of β-secondary structure rich amyloid fibrils in vivo. Here we show that full-length apoA-I IOWA has a decreased lipid binding capability, an increased amino terminal sensitivity to protease, and a propensity to form annular protofibrils visible by electron microscopy. The molecular basis for the conversion of apolipoprotein A-I to a pro-amyloidogenic form was examined by electron paramagnetic resonance spectroscopy. Our recent findings [Lagerstedt, J. O., Budamagunta, M. S., Oda, M. N., and Voss, J. C. (2007) J Biol Chem, 282,[9143][9144][9145][9146][9147][9148][9149] indicate that Gly26 in native apo-protein separates a preceding β-strand structure (residues 20-25) from a downstream largely α-helical region. The current study demonstrates that the G26R variant promotes a structural transition of positions 27-56 to a mixture of coil and β-strand secondary structure. Microscopy and staining by amyloidophilic dyes suggest that this alteration extends throughout the protein within one week of incubation in vitro, leading to insoluble aggregates of distinct morphology. The severe consequences of the Iowa mutation likely arise from the combination of losing the contribution of the native Gly residue in terminating β-strand propagation and the promotion of β structure when an Arg is introduced adjacent to succeeding residue of identical charge and size, Arg27.Apolipoprotein A-I (apoA-I) 1 is the primary protein component of high density lipoprotein (HDL), where it plays a key role in reverse cholesterol transport, a primary mediator of cholesterol efflux and phospholipid metabolism. In reverse cholesterol transport, apoA-I interacts with several members of this pathway, including ATP-binding cassette transporters (ABCA1, ABCG1, and ABCG4), lecithin:cholesterol acyltransferase (LCAT), and † This work was supported by National Institutes of Health grants HL77268, HL78615 and HL073826-01, and by the American Heart Association Scientist Development Grant 0235222N. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR-12088-01 from the National Center (1) ]. The conformational adaptability of apoA-I (2),(3, 4) facilitates the processing of HDL by these distinct receptors and enzymes. For example with lipid binding, apoA-I undergoes a substantial change in structure, wherein the apoA-I helix bundle unfurls into an extended alpha helical "looped belt" conformation that resides on the periphery of the lipid particle [reviewed in (3, 5)]. This structural plasticity has made apoA-I difficult to examine and only recently has detailed structural information become available. Notably, the crystal structure of lipid-free apoA-I provides important new insight into the organization of the lipid-free protein, illuminating a N-terminal four-helix bundle followed by a more flexible C-terminal domain (6). However, features of the structure, parti...

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na The ubiquitous plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) 4 plays central roles in cellular pH and volume homeostasis, cell migration, proliferation, and survival, and increased NHE1 activity contributes to ischemia-reperfusion injury as well as tumor growth and proliferation (1, 2). Hence, the ability to selectively block NHE1, although of high clinical relevance, is hampered by a lack of detailed understanding of NHE1 structure and mechanism of ion translocation. Hydropathy analyses and accessibility studies indicate that NHE1 has 12 transmembrane (TM) segments and a large Cterminal cytoplasmic region (3). Cysteine accessibility studies suggest the presence of two small re-entrant loops between TM IV and TM V (intracellular loop (IL) II) and TM VIII and TM IX (IL IV), respectively, and a larger re-entrant loop between TM IX and TM X (extracellular loop V) (1, 3). Portions of IL II and IL IV are located within the membrane and accessible from either side of the membrane, suggesting that they may form structures lining an aqueous pore and could be involved in ion translocation by NHE1 (3). Extracellular loop V is also interesting in this regard, because it resembles the Ploops found in voltage-gated ion channels (1, 3). These putative re-entrant loops are highly conserved among several NHE1 homologs, consistent with the notion that they are critical for NHE1 function (3-5).A number of regions within the NHE1 protein have been implicated in inhibitor binding, e.g. TM IV and TM IX (6 -11); however, the mechanism(s) of interaction between NHE1 and its commonly used inhibitors, amiloride and benzoyl guanidine type compounds, remain to be fully elucidated.Using a comparative approach based on chimeras generated using human NHE1 (hNHE1) and two NHE1 homologs (flounder paNHE1 and Amphiuma tridactylum NHE1) with high sequence homology to hNHE1 yet markedly different inhibitor profiles (4, 5), we previously obtained novel information on the regions of NHE1 important for inhibitor binding and ion transport (12). These studies confirmed that TM IV plays a central role in inhibitor binding (12) as suggested by earlier point mutation studies (6 -11). Moreover, we demonstrated that regions in TM X-XI and/or IL V and extracellular loop VI are important determinants of inhibitor sensitivity (12).The three-dimensional structure of NHE1 is unknown; however, the structure of the distantly related bacterial (Esch-