Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate

Two N-acetylgalactosaminyltransferases, designated I and II, have been purified from the microsomal fraction of calf arterial tissue and separated on Bio-Gel A. N-Acetylgalactosaminyltransferase I was purified 450-fold. It requires Mn2+ for maximal activity and transfers N-acetylgalactosamine residues from UDP-[1-3H]GalNAc in beta-glycosidic configuration to the non-reducing terminus of the acceptor substrates GlcA(beta 1-3)Gal(beta 1-3)Gal, GlcA(beta 1-3)Gal(beta 1-4)Glc and GlcA(beta 1-3)Gal. Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase II, which transfers N-acetylgalactosamine from UDP-[1-3H]GalNAc to the non-reducing glucuronic acid residues of oligosaccharide acceptor substrates. Maximum transfer rates were obtained with a decasaccharide derived from chondroitin. Longer or shorter-chain chondroitin oligosaccharides are less effective acceptor substrates. All reaction products formed by N-acetylgalactosaminyltransferases I and II are substrates of beta-N-acetylhexosaminidase, which splits off the transferred [1-3H]GalNAc completely. In the microsomal fraction N-acetylgalactosaminyltransferase II had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme II loses much of its activity during the purification procedure and undergoes rapid thermodenaturation. GlcA-Gal-Gal is a characteristic sequence of the carbohydrate-protein linkage region of proteochondrioitin sulfate. The acceptor capacity of this trisaccharide suggests that N-acetylgalactosaminyltransferase I is involved in the synthesis of the carbohydrate-protein linkage region. Since N-acetylgalactosaminyltransferase II is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.     

Heparan sulfate proteoglycans are involved in opiate receptor-mediated cell migration

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.     

Serines 890 and 896 of the NMDA receptor subunit NR1 are differentially phosphorylated by protein kinase C isoforms

The NR1 subunit of the NMDA receptor has two serines (S890 and S896) whose phosphorylation by protein kinase C (PKC) differentially modulates NMDA receptor trafficking and clustering. It is not known which PKC isoforms phosphorylate these serines. In primary cultures of cerebellar neurons, we examined which PKC isoforms are responsible for the phosphorylation S890 and S896. We used specific inhibitors of PKC isoforms and antibodies recognizing specifically phosphorylated S890 or S896. The results show that PKC alpha phosphorylates preferentially S896 and PKC gamma preferentially S890. Activation of type I metabotropic glutamate receptors (mGluRs) with DHPG (3,5-dihyidroxy-phenylglycine) activates PKC gamma but not PKC alpha or beta. We found that activation of mGluRs by DHPG increases S890 but not S896 phosphorylation, supporting a role for PKC gamma in the physiological modulation of S890 phosphorylation. It is also shown that the pool of NR1 subunits present in the membrane surface contains phosphorylated S890 but not phosphorylated S896. This supports that differential phosphorylation of S890 and S896 by different PKC isoforms modulates cellular distribution of NMDA receptors and may also contribute to the selective modulation of NMDA receptor function and intracellular localization.     

The Spir actin organizers are involved in vesicle transport processes

The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.     

Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.     

PINK1 Kinase Catalytic Activity Is Regulated by Phosphorylation on Serines 228 and 402

Mutations in the PINK1 gene cause early-onset recessive Parkinson disease. PINK1 is a mitochondrially targeted kinase that regulates multiple aspects of mitochondrial biology, from oxidative phosphorylation to mitochondrial clearance. PINK1 itself is also phosphorylated, and this might be linked to the regulation of its multiple activities. Here we systematically analyze four previously identified phosphorylation sites in PINK1 for their role in autophosphorylation, substrate phosphorylation, and mitophagy. Our data indicate that two of these sites, Ser-228 and Ser-402, are autophosphorylated on truncated PINK1 but not on full-length PINK1, suggesting that the N terminus has an inhibitory effect on phosphorylation. We furthermore establish that phosphorylation of these PINK1 residues regulates the phosphorylation of the substrates Parkin and Ubiquitin. Especially Ser-402 phosphorylation appears to be important for PINK1 function because it is involved in Parkin recruitment and the induction of mitophagy. Finally, we identify Thr-313 as a residue that is critical for PINK1 catalytic activity, but, in contrast to previous reports, we find no evidence that this activity is regulated by phosphorylation. These data clarify the regulation of PINK1 through multisite phosphorylation.     

The human renal sodium sulfate cotransporter (SLC13A1; hNaSi-1) cDNA and gene: organization, chromosomal localization, and functional characterization

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have determined the structure of the human Na+-sulfate cotransporter (hNaSi-1) cDNA (Human Genome Nomenclature Committee-approved symbol SLC13A1) and gene (NAS1). hNaSi-1 encodes a protein of 595 amino acids with 13 putative transmembrane domains. hNaSi-1 mRNA expression was exclusive to the human kidney. Expression of hNaSi-1 protein in Xenopus oocytes demonstrated a high-affinity Na+-sulfate cotransporter that was inhibited by selenate, thiosulfate, molybdate, tungstate, citrate, and succinate. Antisense inhibition experiments suggest hNaSi-1 to represent the major Na+-sulfate cotransporter in the human kidney. NAS1 was localized on human chromosome 7, mapped to 7q31-q32, near the sulfate transporter genes, DRA and SUT-1. The NAS1 gene contains 15 exons, spanning over 83 kb in length. Knowledge of the structure, function, and chromosomal localization of hNaSi-1 will permit the screening of NAS1 mutations in humans with disorders in renal sulfate reabsorption and homeostasis.     

Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.     

Genetics of sulfate transport by Salmonella typhimurium

Sixty-four mutants were isolated from the LT-2 wild-type strain of Salmonella typhimurium by selecting for chromate resistance. The majority of lesions were shown to lie in the cysA gene. (i) The mutants cannot take up sulfate, a finding which verifies the role of cysA in sulfate transport. In addition, 52 sulfate-transport mutants isolated without chromate selection were defective in the cysA gene. (ii) Most had less than 25% of the binding activity of the wild-type strain. (iii) Most had normal sulfite reductase (H(2)S-nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.8.1.2) activity. (iv) Their sulfate-binding protein (binder) appears electrophoretically and immunologically normal. (v) Amber cysA mutants also make apparently normal binder in small amounts. (vi) All classical cysA mutants tested, including two with long deletions, had normal binding activity. From these observations, it is suggested that the cysA gene does not code for the binder. But many mutations in this gene reduce the binding activity in some unknown way. Other mutants, identified as cysB mutants, had neither binding nor uptake activities and their sulfite reductase activities were similarly reduced, thus confirming the regulatory role of the cysB gene. When binder was detectable, it had wild-type properties. No mutations in the binder gene were found among more than 100 mutants examined. Thus, sulfate binding has not been established as a part of sulfate transport. However, the production of binder is intimately connected with cysA, the established sulfate transport gene, and is regulated by the same mechanism that regulates both transport and the rest of the cysteine biosynthetic pathway.     

Cytosolic Hsp70s are involved in the transport of aminopeptidase 1 from the cytoplasm into the vacuole

Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.     

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity

Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K(m) values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-β1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation.     

Regulation of sulfate transport in neurospora by transinhibition and by inositol depletion

Neurospora possesses two distinct sulfate transport systems, a low-affinity form (Permease I) which is the only type found in conidia, and a second species (Permease II) which predominates during the mycelial stage. Although methionine represses the synthesis of both of these permeases, inorganic sulfate only partially represses the mycelial form and does not affect the synthesis of Permease I. Both transport systems are also regulated by transinhibition. The transinhibition which occurs in mycelia is not due to an intracellular pool of inorganic sulfate, but is instead exerted by an early intermediate of the sulfate assimilatory pathway.The development of functional sulfate transport activity depends upon genetic and metabolic events which affect the cell membrane. The synthesis of sulfate permease activity in the inos mutant requires an exogenous supply of inositol. The effect of the cot mutant, which is thought to interfere with membrane synthesis, also prevents the development of sulfate permease at the restrictive temperature. The maintenance of pre-existing functional sulfate permease activity apparently also requires a continuous renewal of membrane components since withdrawal of inositol from inos mutants results in a rapid inactivation of transport activity.     

Thymosin beta 4 expression and nuclear transport are regulated by hMLH1

For hMLH1, a key enzyme of DNA mismatch repair and frequently mutated in human cancers, several additional functions have been suggested. We now identified Thymosin β₄ (Tβ₄), an actin-binding and cell motility regulating protein, by bacterial two-hybrid screening. Interaction was confirmed by coimmunoprecipitation. Tβ₄ was weakly expressed in the hMLH1-deficient cell lines 293T and HCT-116. Reconstitution of hMLH1 resulted in strong expression of Tβ₄. Confocal laser microscopy revealed nuclear colocalization of both proteins. Reconstitution with hMLH1 mutants lacking a functional nuclear localization sequence resulted in cytoplasmatic retention of both proteins. After Tβ₄− or hMLH1-siRNA treatment, cell migration of hMLH1-proficient cells was markedly decreased.Our results show that hMLH1 interacts with Tβ₄ and regulates its expression and nuclear transport. Moreover, loss of hMLH1 causes Tβ₄ deprivation and results in reduced migratory activity in vitro. These data give insight into novel functions of hMLH1 and probably disease related dysregulated mechanisms.     

Molybdate transport through the plant sulfate transporter SHST1

Molybdenum is an essential micronutrient required by plants. The mechanism of molybdenum uptake in plants is poorly understood, however, evidence has suggested that sulfate transporters may be involved. The sulfate transporter from Stylosanthes hamata, SHST1, restored growth of the sulfate transport yeast mutant, YSD1, on media containing low amounts of molybdate. Kinetic analysis using 99MoO4(2-) demonstrated that SHST1 enhanced the uptake of molybdate into yeast cells at nM concentrations. Uptake was not inhibited by sulfate, but sulfate transport via SHST1 was reduced with molybdate. These results are the first measurement of molybdate transport by a characterised plant sulfate transport protein.     

Ammonium transport and CitAMT1 expression are regulated by light and sucrose in Citrus plants

Here the isolation and characterization of CitAMT1 cDNA from citrange Troyer (Citrus sinensis L. OsbeckxPoncirus trifoliata Blanco) is reported, suggesting that this belongs to the AMT gene family, which is involved in the high-affinity transport system (HATS). Results show that in Citrus plants, the HATS is much more dependent on the light conditions and C status of the roots than the low-affinity transport system. Most importantly, a strong correlation was found between the regulation of both HATS activity and CitAMT1 expression. CitAMT1 expression is sucrose-stimulated and may account for the regulation of NH(4)(+) HATS. Furthermore, a similar link was also recorded with photosynthetic activity in the shoots, suggesting that the variations in production and transport of photosynthates to the roots are responsible for the diurnal changes of both CitAMT1 expression and NH(4)(+) HATS activity. On the other hand, results indicate that the effect of stimulating light on CitAMT1 expression and NH(4)(+) HATS activity is independent of the circadian rhythm. Finally, CitAMT1 expression seems to be specifically stimulated by sucrose, suggesting that sucrose is a pivotal signal governing both assimilate partitioning from source organs and assimilate utilization in sink organs.     

Mechanism of sulfate transport inhibition by cycloheximide in plant tissues

Inhibition by cycloheximide of sulfate transport in both barley roots (Hordeum vulgare L.) and potato tuber (Solanum tuberosum L.) increases with increasing inhibitor concentration only to a limited extent, depending on the length of the tissue incubation with the inhibitor. In contrast to this, increasing concentrations of dinitrophenol have a rapid and total inhibitory effect on the active transport. Leucine transport in the same tissues is strongly inhibited by dinitrophenol but is not affected by cycloheximide, whereas incorporation into protein is mainly inhibited by cycloheximide. It appears that the mechanism of transport inhibition by cycloheximide in plant tissues consists in stopping new carrier synthesis and not in the disruption of energy flow. Sulfate carriers show comparable decay rates in barley roots and potato tuber, the mean life being shorter than that of the leucine carriers. These appear more stable in roots than in storage tissues.