Mutation of Asparagine 76 in the Center of Glutamine Transporter SNAT3 Modulates Substrate-induced Conductances and Na+ Binding

The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (γ-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na⁺-glutamine cotransport-H⁺ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na⁺-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na⁺. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na⁺ -binding site.The amino acid and auxin permease superfamily comprises a wide variety of transport proteins. In mammals, three distinct solute carrier families (SLC) belong to this superfamily, namely SLC32, SLC36, and SLC38 (1). Despite belonging to the same superfamily, the three solute carrier families have different transport mechanisms. The SLC32 family has only one member, the vesicular inhibitory amino acid transporter, which supposedly carries out a H⁺-GABA (γ-aminobutyric acid) antiport (2). The SLC36 family comprises four members, two of which have been characterized in more detail. These are the proton amino acid cotransporters 1 and 2 (PAT1 and 2) that carry out glycine and proline uptake in kidney and intestine and are mutated in iminoglycinuria (3, 4). The SLC38 family is comprised of 11 members, 5 of which have been characterized in more detail (5). Two different transport mechanisms are found within this family, namely the Na⁺-amino acid cotransporters SNAT1, SNAT2, and SNAT4 and the Na⁺-amino acid cotransporters-H⁺-antiporters SNAT3 and SNAT5. Transporters of the superfamily play a key role in inhibitory and excitatory neurotransmission, metabolite absorption, and liver metabolism. Despite their important roles in mammalian physiology, relatively little is known about the structure and function of these transporters.The activity of ion-coupled membrane transporters is frequently associated with currents which de- or hyperpolarize the cell membrane. These currents may be due to electrogenic transport stoichiometry and/or to a non-stoichiometric ion conductance (6). Transport-associated ion conductances have been identified in a number of transporters but have been particularly well studied in several Na⁺-coupled neurotransmitter transporters (7–11). Transport-associated conductances have also been observed in electroneutral transporters that do not carry out net charge movement (8, 12–15). The glutamine transporter SNAT3, for instance, has a transport mechanism in which glutamine uptake is coupled to the cotransport of 1Na⁺ and the antiport of 1H⁺ and, hence, is unaffected by changes of the membrane potential (13, 16). Despite the electroneutral transport mechanism, substrate uptake is accompanied by inward currents, which are carried by cations below pH 7 and by protons at alkaline pH. In addition, a substrate-independent cation conductance and a Na⁺/H⁺ exchange activity has been observed (17). Non-stoichiometric currents can be mediated by the same ions that are involved in the coupled transport process, such as in the case of SNAT3, but may also be carried by different ions. Stoichiometric glutamate transport, for instance, involves Na⁺, H⁺, and K⁺ ions, whereas the glutamate transport-associated conductance is carried by chloride (18).A crucial question concerning transporter-associated ion conductances is whether the conducting pore coincides with the translocation pathway of the substrate and whether both use the same critical residues. In the case of the glutamate transporters, evidence has been presented suggesting that different residues are critical for the anion conductance than for substrate transport (19, 20) but that they all line the same pathway (21). Here we show that asparagine 76 of SNAT3 is critical for substrate-induced ion conductance and affects binding of the cosubstrate Na⁺. In addition we show that this residue is likely to be localized in the translocation pore in the center of the membrane.     

Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Human concentrative nucleoside transporter, hCNT3, mediates Na⁺/nucleoside and H⁺/nucleoside co-transport. We describe a new approach to monitor H⁺/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H⁺-coupled nucleoside transport, pH 5.5, under both Na⁺-free and Na⁺-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H⁺-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H⁺ flux) in the presence or absence of Na⁺ (1.09 ± 0.11 or 1.18 ± 0.32 mm min⁻¹, respectively). This suggests that in acidic Na⁺-containing conditions, 1 Na⁺ and 1 H⁺ are transported per uridine molecule, while in acidic Na⁺-free conditions, 1 H⁺ alone is transported/uridine. In acid environments, including renal proximal tubule, H⁺/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H⁺-coupled transport proteins.Nucleosides are hydrophilic molecules that require transport proteins to mediate their movement across the plasma membrane (1). Human (h)⁷ nucleoside transport (NT) proteins catalyze the vectorial transport of nucleosides, using either concentrative (C) or equilibrative (E) mechanisms (2). hCNTs use either a Na⁺ or H⁺ gradient to accumulate nucleosides against their concentration gradient, whereas hENTs mediate facilitated diffusion of nucleosides down their concentration gradient (3). Nucleoside transporters also transport anti-cancer and anti-viral drugs, and cellular expression of nucleoside transporters is important in cancer therapy as well as in the treatment of cardiovascular, parasitic, and viral diseases (4, 5).Members of the SLC28 family of concentrative nucleoside transporters (CNTs) divide into two phylogenetic subfamilies: hCNT1/2 belonging to one subfamily, and hCNT3 to the other (6–8). Cation substitution and charge/flux ratio studies suggest that hCNT1/2 couple the inward movement of nucleoside to the Na⁺ electrochemical gradient with a 1:1 stoichiometry, whereas hCNT3 can couple nucleoside transport to either the Na⁺ gradient (2 Na⁺:1 nucleoside) or a H⁺ gradient (1 H⁺:1 nucleoside) in the absence of Na⁺ (9, 10). The 2:1 coupling ratio of hCNT3 allows it to develop a trans-membrane nucleoside concentration gradient up to 10-fold higher than that of hCNT1 or hCNT2 (9, 11). At pH 5.5, hCNT3 also transports uridine in the presence of Na⁺ with a 2 cation:1 nucleoside stoichiometry, which raises the possibility that 1 H⁺ and 1 Na⁺ may be transported per nucleoside molecule in these conditions (9–12). Up to this point, however, there has been no direct demonstration that hCNT3 can transport H⁺.Concentrative nucleoside transport has previously been investigated using the Xenopus laevis oocyte expression system and both electrophysiology (two-microelectrode voltage clamp technique) and radioisotope flux measurements (6–9, 12). Electrophysiological experiments are advantageous in that they measure the current induced by addition of substrate in real-time, but they are time-consuming and require specialized equipment and skills. Radioisotope flux assays measure the accumulation of radiolabeled substrate. The need for radiolabeled substrate restricts the range of permeants able to be studied. In addition, radioisotope flux assays are not done in real-time and are labor-intensive, requiring large numbers of oocytes.An attractive alternative approach for the study of hCNT3 would be to measure pH in the immediate vicinity of its intracellular face during H⁺/nucleoside co-transport. These measurements could take advantage of the remarkable progress achieved in the development of genetically encoded fluorophores (13). Indeed, all members of the extended family of homologues and variants of the Aequorea victoria green fluorescent protein (avGFP) exhibit pH-dependent changes in their fluorescent intensity. The spectral changes that occur upon a change in pH can be intensiometric (14), excitation ratiometric (14), emission ratiometric (15), or both excitation and emission ratiometric (16). The apparent pK_a (pK_a′, equal to the pH at which the fluorescence is half-maximal in intensity) for a specific fluorescent protein (FP) is acutely dependent on specific amino acid substitutions in close proximity to the chromophore and can range from less than 3 (17, 18) to greater than 8 (19). Variants with pK_a′ values that are relatively close to intracellular pH values (i.e. ∼7.3 for the mammalian cytosol (20)) are particularly useful as genetically encoded biosensors for dynamic measurement of proton concentrations in living cells.A major development in the area of FP technology has been the identification (21) and subsequent optimization (22, 23) of red fluorescent protein (RFP) homologues of avGFP. The first (monomeric RFP 1 (mRFP1)) (23) and second (the mFruit series) (22) generation-optimized RFPs, derived from tetrameric Discosoma RFP (21), suffer from relatively low brightness relative to other common hues of FP. For example, of the three most red-shifted second generation mFruit variants (mTangerine, mStrawberry, and mCherry) (22), the brightest (mStrawberry) has only 44% of the intrinsic brightness (proportional to the product of extinction coefficient (ϵ) and quantum yield (Φ)) of the popular yellow FP (YFP) Citrine (24) and 76% of the brightness of enhanced avGFP. This limitation has been partially addressed by third generation mRFPs, specifically mApple and TagRFP-T, with fluorescent brightness values on par with, or better than, that of enhanced avGFP (25).Generally speaking, the most red-shifted RFPs derived from Discosoma RFP are relatively pH-insensitive, with the majority of variants having pK_a′ values < 5 (22, 25). A notable exception is the recently reported mApple variant with a pK_a′ of 6.5 (25). The more blue-shifted of the mFruit variants (i.e. mOrange) also have pK_a′ values of 6.5 (22). Several variants of mRFP1 with pK_a′ values >7.5 have been previously reported (26).Here we report the engineering of a pH-sensitive mFruit variant through multiple rounds of directed evolution by random mutagenesis. This RFP, called mNectarine, is appropriate to measure physiological pH changes in mammalian cells, because it has a pK_a′ of 6.9. We have developed a new method to measure H⁺/nucleoside co-transport in mammalian cells, which utilizes hCNT3''s H⁺ coupling characteristics and the pH sensitivity of mNectarine. We fused mNectarine to the cytosolic N terminus of hCNT3 to generate mNect.hCNT3. Fusion of the fluorescent H⁺ sensor to hCNT3 enabled measurement of pH at the intracellular surface of hCNT3, and provided insight into the mechanism of hCNT3 H⁺/uridine co-transport.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na⁺-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na⁺ and/or H⁺ electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C−, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11–13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F).Specialized nucleoside transporter proteins are required for passage of nucleosides and hydrophilic nucleoside analogs across biological membranes. Physiologically, nucleosides serve as nucleotide precursors in salvage pathways, and pharmacologically nucleoside analogs are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases (1, 2). Additionally, adenosine modulates numerous cellular events via purino-receptor cell signaling pathways, including neurotransmission, vascular tone, immune responses, and other physiological processes (3, 4).Human nucleoside transporter proteins are divided into two families: the SLC29 ENT (equilibrative nucleoside transporter) family and the SLC28 CNT (concentrative nucleoside transporter) family (3, 5–7). hENTs³ mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types (8). Additionally, the hENT2 isoform is capable of nucleobase transport (9). hCNTs, in contrast, are inwardly directed Na⁺-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types (10, 11). hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, and couple Na⁺/nucleoside cotransport with 1:1 stoichiometry (12–18). In contrast, hCNT3 is broadly selective for both pyrimidine and purine nucleosides and couples Na⁺/nucleoside cotransport with 2:1 stoichiometry (10, 18, 19). hCNT3 is also capable of H⁺/nucleoside cotransport with a coupling stoichiometry of 1:1, whereby one of the two Na⁺ binding sites also functionally interacts with H⁺ (18, 19).Current models of CNT topology have 13 putative transmembrane helices (TMs) (10, 14, 16, 20). Two additional TMs (designated 5A and 11A) are weakly predicted by computer algorithms (20), and immunocytochemical experiments with site-specific antibodies and studies of native and introduced glycosylation sites have confirmed an intracellular N terminus and an extracellular C terminus (20). Chimeric studies involving hCNTs and hfCNT, a CNT from the ancient marine prevertebrate, the Pacific hagfish Eptatretus stouti, have revealed that the functional domains responsible for CNT nucleoside selectivity and cation coupling reside within the C-terminal TM 7–13 half of the protein (19, 21). NupC, an H⁺-coupled CNT family member from Escherichia coli, lacks TMs 1–3 but otherwise shares a topology similar to that of its eukaryote counterparts (22, 23).A functional cysteineless version of hCNT3 has been generated by mutagenesis of endogenous cysteine residues to serine, resulting in the cysteineless construct hCNT3C− employed originally in a yeast expression system for substituted cysteine accessibility method (SCAM) analysis of TMs 11, 12, and 13 using methanethiosulfonate (MTS) reagents (24). Subsequently, we have also characterized hCNT3C− in the Xenopus oocyte expression system (25) and have initiated SCAM analyses with the alternative thiol-specific reagent p-chloromercuribenzene sulfonate (PCMBS) (26). Measured by transport inhibition, reactivity of introduced cysteine residues with PCMBS, which is both membrane-impermeant and hydrophilic, indicates pore-lining status and access from the extracellular medium; the ability of a permeant to protect against this inhibition denotes location within, or closely adjacent to, the permeant-binding pocket (27, 28). Continuing the investigation of hCNT3 C-terminal membrane topology and function, the present study reports results of PCMBS SCAM analyses of TMs 11–13, including loop regions linking the putative TMs not previously studied using MTS reagents.In earlier structure/function studies of hCNT3, we identified a cluster of conformationally sensitive residue positions in TM 12 (Ile⁵⁵⁴, Tyr⁵⁵⁸, and Cys⁵⁶¹) that exhibit H⁺-activated inhibition by PCMBS, with uridine protection evident for Tyr⁵⁵⁸ and Cys⁵⁶¹ (26). Located deeper within the plane of the membrane, other uridine-protectable residue positions in TM 12 were PCMBS-sensitive in both H⁺- and Na⁺-containing media (26). hCNT3 Glu⁵¹⁹ and the corresponding residue in hCNT1 (Glu⁴⁹⁸) in region TM 11A were also identified as having key roles in permeant and cation binding and translocation (29, 30), and hCNT3 E519C showed inhibition of uridine uptake by PCMBS (30). Centrally positioned within the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F), residue 519 is proposed to be a direct participant in cation coupling via the common hCNT3 Na⁺/H⁺-binding site that, in other CNTs, is either Na⁺-specific (e.g. hCNT1) or H⁺-specific (e.g. NupC) (30).Building upon the prior work with MTS reagents and other structure/function studies of hCNT3, the present study identified new residues of functional importance in the C-terminal one-third of hCNT3, established the orientations and α-helical structures of TMs 11–13, and determined a novel membrane-associated topology for the TM 11A region of the protein. A revised CNT membrane architecture is proposed.     

Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica

Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.Pyridine nucleotide transhydrogenase (PNT) participates in the bioenergetic processes of the cell. PNT generally resides on the cytoplasmic membranes of bacteria and the inner membrane of mammalian mitochondria (3, 16) and utilizes the electrochemical proton gradient across the membrane to drive NADPH formation from NADH (14, 15, 39) according to the reaction H⁺out + NADH + NADP⁺↔H⁺in + NAD⁺ + NADPH, where “out” and “in” denote the cytosol and the matrix of the mitochondria, or the periplasmic space and the cytosol of bacteria, respectively.PNT has been identified in several protozoan parasites, including Entamoeba histolytica (8, 51), Eimeria tenella (17, 47), Mastigamoeba balamuthi (11) Plasmodium falciparum (10), Plasmodium yoelii (6), and Plasmodium berghei (12). In general, PNT contains conserved structural units consisting of three domains, the NAD(H)-binding domain (domain I [dI]) and the NADP(H)-binding domain (domain III [dIII]), both of which face the matrix side of the eukaryotic mitochondria or the cytoplasmic side in bacteria, and the hydrophobic domain (domain II [dII]), containing 11 to 13 transmembrane regions. PNT from E. tenella and E. histolytica exists as a single polypeptide in an unusual configuration consisting of dIIb-dIII-dI-dIIa, with a 38-amino-acid-long linker region between dIII and dI (48).E. histolytica, previously considered an “amitochondriate” protist, is currently considered to possess a mitochondrion-related organelle with reduced and divergent functions, the mitosome (1, 21, 23a, 26, 42). Our recent proteomic study of isolated mitosomes identified about 20 new constituents (26), together with four proteins previously demonstrated in E. histolytica mitosomes: Cpn60 (8, 19, 21, 42), Cpn10 (46), mitochondrial Hsp70 (2, 44), and mitochondrion carrier family (MCF) (ADP/ATP transporter) (7). Despite the early presumption of PNT being localized in mitosomes (8), based on the amino-terminal region rich in hydroxylated (five serines and threonines) and acidic (three glutamates) amino acids, which slightly resembles known mitochondrion- and hydrogenosome-targeting sequences (8, 35), PNT was not discovered in the mitosome proteome. We also doubted this premise because PNT was one of the major proteins identified in isolated phagosomes (32, 33). Thus, the intracellular localization and trafficking of PNT remain unknown.In this report, we showed that E. histolytica PNT (EhPNT) is localized to various vesicles and vacuoles, including lysosomes and phagosomes, using wild-type amoebae and antiserum raised against recombinant EhPNT and an E. histolytica line expressing EhPNT with a carboxyl-terminal hemagglutinin (HA) epitope tag and anti-HA antibody. We also showed that all domains of EhPNT are required for its trafficking to the acidic compartment by using amoeba transformants expressing the HA tag or green fluorescent protein (GFP) fused with a region containing various domains of EhPNT.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Heteromeric AtKC1��AKT1 Channels in Arabidopsis Roots Facilitate Growth under K+-limiting Conditions

Plant growth and development is driven by osmotic processes. Potassium represents the major osmotically active cation in plants cells. The uptake of this inorganic osmolyte from the soil in Arabidopsis involves a root K⁺ uptake module consisting of the two K⁺ channel α-subunits, AKT1 and AtKC1. AKT1-mediated potassium absorption from K⁺-depleted soil was shown to depend on the calcium-sensing proteins CBL1/9 and their interacting kinase CIPK23. Here we show that upon activation by the CBL·CIPK complex in low external potassium homomeric AKT1 channels open at voltages positive of E_K, a condition resulting in cellular K⁺ leakage. Although at submillimolar external potassium an intrinsic K⁺ sensor reduces AKT1 channel cord conductance, loss of cytosolic potassium is not completely abolished under these conditions. Depending on channel activity and the actual potassium gradients, this channel-mediated K⁺ loss results in impaired plant growth in the atkc1 mutant. Incorporation of the AtKC1 subunit into the channel complex, however, modulates the properties of the K⁺ uptake module to prevent K⁺ loss. Upon assembly of AKT1 and AtKC1, the activation threshold of the root inward rectifier voltage gate is shifted negative by approximately −70 mV. Additionally, the channel conductance gains a hypersensitive K⁺ dependence. Together, these two processes appear to represent a safety strategy preventing K⁺ loss through the uptake channels under physiological conditions. Similar growth retardation phenotypes of akt1 and atkc1 loss-of-function mutants in response to limiting K⁺ supply further support such functional interdependence of AKT1 and AtKC1. Taken together, these findings suggest an essential role of AtKC1-like subunits for root K⁺ uptake and K⁺ homeostasis when plants experience conditions of K⁺ limitation.Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K⁺ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ∼20 μm and a low affinity transport system with K_m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K⁺ channels AKT1·AtKC1 and members of the HAK·KT·KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5–9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K⁺ channel family. AKT1 is a voltage-dependent inward-rectifying K⁺ channel mediating potassium uptake over a wide range of external potassium concentrations (10–15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K⁺ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 μm and less) (11, 12, 15). Rescue of yeast growth on 20 μm K⁺ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K⁺ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with AKT1 (18–20). AtKC1 α-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K⁺ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 α-subunits do not form K⁺ channels per se but modulate the properties of the AKT1·AtKC1 heterocomplex (20–22). Previously, two groups in their ground-breaking studies demonstrated that AKT1 is activated by the CBL²-interacting, serine/threonine kinase, CIPK23, particularly under low K⁺ conditions (23, 24). CIPK23 itself was shown to be activated by the two calcineurin B-like proteins, CBL1 and 9, acting in a Ca²⁺-dependent manner upstream of CIPK23 (25, 26). Genetic disruption of these elements resulted in transgenic plants exhibiting a phenotype comparable with that of the AKT1 loss of function mutant. This regulatory system, based on a calcium sensor, a protein kinase, and a K⁺ channel, was functionally reconstituted in Xenopus oocytes (23, 24, 27), suggesting that these elements are essential and sufficient to operate as a low K⁺-sensitive potassium uptake system. Here we report on the physiological properties of the heteromeric K⁺ uptake module formed by the predominant root potassium uptake channel subunits, AKT1 and AtKC1 and its regulating kinase complex, CBL1 and CIPK23. Our studies show that the physical interaction of the CBL1·CIPK23 complex is specific for AKT1 channels and does not involve the AtKC1 subunit. AKT1 possesses a K⁺ (absence) sensor affecting channel activity at submillimolar K⁺ concentrations by strongly reducing its maximal cord conductance. Despite this K⁺ sensor, upon activation, AKT1 homomeric channels were shown to represent a potassium leak at low external potassium concentrations. Integration of AtKC1 into the K⁺ uptake module, however, prevented potassium loss by modulating both the voltage sensor and conductance in the channel complex. Moreover, activation of the AKT1-like maize channel ZMK1 by CBL1·CIPK23 suggests a conserved interaction and regulation across monocot and dicotyledonous plant species. Our biophysical studies as well as growth assays with plant mutant lines lacking the respective channels underline that acquisition of potassium under limiting K⁺ conditions is mediated via the root AKT1·AtKC1 K⁺ uptake channel complex.