The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus

It has been suggested that ZIP7 (Ke4, Slc39a7) belongs to the ZIP family of zinc transporters. Transient expression of the V5-tagged human ZIP7 fusion protein in CHO cells led to elevation of the cytoplasmic zinc level. However, the precise function of ZIP7 in cellular zinc homeostasis is not clear. Here we report that the ZIP7 gene is ubiquitously expressed in human and mouse tissues. The endogenous ZIP7 was associated with the Golgi apparatus and was capable of transporting zinc from the Golgi apparatus into the cytoplasm of the cell. Moreover, by using the yeast mutant strain Deltazrt3 that was defective in release of stored zinc from vacuoles, we found that ZIP7 was able to decrease the level of accumulated zinc and in the meantime to increase the nuclear/cytoplasmic labile zinc level in the ZIP7-expressing zrt3 mutant. We showed that the protein expression of ZIP7 was repressed under zinc-rich condition, whereas there were no effects of zinc on ZIP7 gene expression and intracellular localization. Neither did zinc deficiency affect the intracellular distribution of ZIP7 in mammalian cells. Our study demonstrates that ZIP7 is a functional zinc transporter that acts by transporting zinc from the Golgi apparatus to the cytoplasm of the cell.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.     

真核生物锌转运体及其活性的调控

真核生物的锌内稳态是由其众多特异转运体协同转运来实现的。有两个锌转运体家族ZIP和CDF被相继发现。ZIP家族的主要功能是摄取锌,而CDF家族成员主要参与锌的外排及锌在细胞内的区室化以达到解毒或贮存的目的。锌可在转录水平和翻译水平调控两类转运体的活性以维持锌在细胞和生物体水平的内稳态。     

Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter

Nucleotide sulfate, namely 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene. We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll). The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins. The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane. The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction. In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart. An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability. It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders. Our findings provide insights into the significance of PAPS transport and post-translational sulfation.     

GDP-fucose uptake into the Golgi apparatus during xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter

The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan alpha,1-2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls.     

Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the spondylocheiro dysplastic Ehlers-Danlos syndrome

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.     

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn²⁺, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca²⁺-uptake transporter thought to support Golgi uptake of Mn²⁺ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca²⁺ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca²⁺ levels. In turn, we found increased cytoplasmic Ca²⁺ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in ⁵⁴Mn²⁺ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca²⁺ and ⁵⁴Mn²⁺ accumulation. While addition of Ca²⁺ positively regulated ZIP-mediated ⁵⁴Mn²⁺ uptake, we show chelation of Ca²⁺ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn²⁺ and other divalent metal ions–mediated ZIP metal transporters.     

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.     

Overexpression of the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells

The canine 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter1 fused to GFP was stably expressed with a typical Golgi localizationin MDCK II cells (MDCK II-PAPST1). The capacity for PAPS uptakeinto Golgi vesicles was enhanced to almost three times thatof Golgi vesicles isolated from untransfected cells. We havepreviously shown that chondroitin sulfate proteoglycans (CSPGs)are several times more intensely sulfated in the basolateralthan the apical secretory pathway in MDCK II cells (Tveit H,Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes differentmodifications en route to the apical and basolateral surfacesof Madin-Darby canine kidney cells. J Biol Chem. 280:29596–29603).Here we demonstrate that increased availability of PAPS in theGolgi lumen enhances the sulfation of CSPG in the apical pathwayseveral times, while sulfation of CSPGs in the basolateral pathwayshows minor changes. Sulfation of heparan sulfate proteoglycansis essentially unchanged. Our data indicate that CSPG sulfationin the apical pathway of MDCK II cells occurs at suboptimalconditions, either because the sulfotransferases involved havehigh K_m values, or there is a lower PAPS concentration in thelumen of the apical secretory route than in the basolateralcounterpart.     

Zinc uptake and radial transport in roots of Arabidopsis thaliana: a modelling approach to understand accumulation

Background and Aims

Zinc uptake in roots is believed to be mediated by ZIP (ZRT-, IRT-like proteins) transporters. Once inside the symplast, zinc is transported to the pericycle, where it exits by means of HMA (heavy metal ATPase) transporters. The combination of symplastic transport and spatial separation of influx and efflux produces a pattern in which zinc accumulates in the pericycle. Here, mathematical modelling was employed to study the importance of ZIP regulation, HMA abundance and symplastic transport in creation of the radial pattern of zinc in primary roots of Arabidopsis thaliana.

Methods

A comprehensive one-dimensional dynamic model of radial zinc transport in roots was developed and used to conduct simulations. The model accounts for the structure of the root consisting of symplast and apoplast and includes effects of water flow, diffusion and cross-membrane transport via transporters. It also incorporates the radial geometry and varying porosity of root tissues, as well as regulation of ZIP transporters.

Key Results

Steady-state patterns were calculated for various zinc concentrations in the medium, water influx and HMA abundance. The experimentally observed zinc gradient was reproduced very well. An increase of HMA or decrease in water influx led to loss of the gradient. The dynamic behaviour for a change in medium concentration and water influx was also simulated showing short adaptation times in the range of seconds to minutes. Slowing down regulation led to oscillations in expression levels, suggesting the need for rapid regulation and existence of buffering agents.

Conclusions

The model captures the experimental findings very well and confirms the hypothesis that low abundance of HMA4 produces a radial gradient in zinc concentration. Surprisingly, transpiration was found also to be a key parameter. The model suggests that ZIP regulation takes place on a comparable timescale as symplastic transport.