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1.
The exact mechanism of transport of boron (B) entering the plant cell as boric acid B(OH)3, has become hotly debated with evidence for both passive and protein facilitated transport. Here we put the controversy to rest by confirming that boron influx into plants can be partially controlled by opening and closing of channel-like transport proteins. Using treatments that were likely to inhibit membrane transporters capable of facilitating B transport, we confirmed that at least 50% of B transport could be contributed by a transporter of some type in barley roots. Based on the physiochemical similarities between B(OH)3 and other solutes that were known to be transported via aquaglyceroporins, we hypothesised that aquaglyceroporins would be likely candidates to facilitate B(OH)3 transport into the cytoplasm. We demonstrated using functional yeast complementation that two barley root aquaglyceroporins, HvPIP1;3 and HvPIP1;4, were both capable of facilitating B transport. This finding has demonstrated yet another function of aquaglyceroporins.Key words: aquaporin, aquaglyceroporin, boron, transport, PIPsThe major intrinsic protein (MIP) superfamily contains aquaporins and the related ‘aquaglyceroporins’ (AQGP), whose numbers and functionality are rapidly expanding.1 These transport proteins are responsible for not only the bidirectional transport of water and glycerol, but also for the transport of other small neutral uncharged solutes. Based on their size, net charge and volume compared to the diameter of the aquaglyceroporin pore, it was predicted that a range of other molecules such as arsenite (AsIII) and silicic acid Si(OH)4 would also permeate aquaglyceroporins, and this has been confirmed.2–5 It has long been argued that because of the strong similarity with H2O, it could reasonably be assumed that H2S would cross membranes via aquaporins. However, it has very recently been demonstrated that membrane fluxes of H2S were insensitive to treatments that inhibited influx of H2O, leading to the conclusion that H2S simply passed through the phospholipid bilayer and not through a protein transporter.6Boron (B), available to plants as boric acid, B(OH)3, can be classed as a small neutral uncharged molecule based on physiochemical similarities to glycerol and arsenite.7 Like H2S, the research surrounding B transport across biological membranes has been highly debated and the literature contains conjecture about the exact mode of transport with evidence for both passive and active transport. Several studies have demonstrated substantial passive B movement through both lipid bilayers and plant membranes, consistent with measurements indicating that B has high lipid solubility which would favor permeation through such membranes.8–12 These data suggested that protein-mediated transport into cells would be redundant and would be short-circuited by the passive leak pathway. However, other reports have indicated that B transport may have an active transport component when plants were grown under B deficient conditions.12,13 The presence of protein-assisted passive transport has proved hard to establish.Our recent work has focused on putting this controversy to rest by attempting to modify B uptake using treatments that should not affect B transfer through the lipid phase of the membrane.7 Firstly, we hypothesized that aquaglyceroporins may be involved in the transport of B and examined influx, efflux and concentration-dependence of B uptake in barley roots using inhibitors known to cause the closure of aquaporins though cytoplasmic acidification (butyric acid) or metabolic inhibition (sodium azide). Results from these experiments demonstrated that a significant component of both B influx and efflux was responsive to these treatments. Metabolic inhibition by sodium azide reduced influx and efflux by 40–50%, while cytoplasmic acidification with butyric acid reduced influx to a lesser but still significant degree.7Secondly, in order to elucidate which transport proteins may be involved, we hypothesised more specifically that the PIP1 subgroup may be able to facilitate the movement of B(OH)3 based on the location of such proteins on the plasma membrane. This had previously been suggested by Dordas et al.14 who showed that a maize aquaporin ZmPIP1 when expressed in Zenopus oocytes could account for at least 25% of B uptake. We selected two aquaglyceroporins isoforms previously characterised from barley roots,15–17 HvPIP1;3 and HvPIP1;4, and functionally expressed these in a Saccharomyces cerevisiae mutant containing a deletion of the yeast native aquaglyceroporin, FPS1. Expression of these PIP1 constructs caused the yeast to become sensitive to B toxicity. Influx measurement revealed that both HvPIP1;3 and HvPIP1;4 were capable of transporting B as indicated by increases of up to 40% in the rate of B uptake. Activation in yeast of some plant Nod 26-like intrinsic proteins (NIPs) that also function as aquaglyceroporins, requires truncation of the N-terminal sequence, presumably because this region contains a control domain. In our yeast experiments, a truncated version of HvPIP1;3 (HvPIP1;3t) was engineered to determine the effect of the removal of the first 44 amino acids from the N-terminal tail on the expression and subsequent B transport capacity. Surprisingly truncation of HvPIP1;3 had little effect on either the expression or transport capacity of HvPIP1;3.As a result of this study it has been firmly established that boron entry into plants can be partially controlled by opening and closing of channel-like transport proteins. Specifically, we have demonstrated that B can be transported via two aquaglyceroporins, HvPIP1;3 and HvPIP1;4. However, we suspect that most of the HvPIP1 subgroup, which contains another 3 members, may all have some capacity to transport B based on high sequence homology amongst the PIP1 subgroup.The confirmation of the ability of PIP1s to transport B contributes greatly to the overall understanding of B transport in the plant system. Recently other aquaglyceroporins NIP5;1 and NIP6;1 have also been shown to be involved in B influx18–20 while a separate class of non-aquaglyceroporins, that are structurally related to anion exchangers, are involved in the active efflux of B under toxicity conditions21,22 or xylem loading under deficiency conditions.23,24Aquaglyceroporins may have evolved to facilitate transport of beneficial and essential nutrients such as Si(OH)4,2 B(OH)3, urea and ammonia25 but other toxic molecules with similar physiochemical characteristics such as AsIII and Sb(OH)3 may have ‘piggy backed’ on the process allowing these toxins to also enter the plant system. An understanding of selectivity mechanism that allows both essential and toxic elements to pass through the aquaglyceroporin pore and into the cytoplasm may have important implications for research into the potential bioremediation of toxic substances. It seems highly probable that other small molecules will be shown to be transported by aquaglyceroporins. There is still much to be learnt about the roles of other classes of MIPs, in particular NIPs, small basic intrinsic proteins (SIPs)26 and tonoplast intrinsic proteins (TIPs) in the movement of these molecules into and within cells. No doubt the roles and functions of aquaglyceroporins within the plant system will continue to grow. 相似文献
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Identification of an H2O2 permeable PIP aquaporin in barley and a serine residue promoting H2O2 transport
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Tomoaki Horie Shizuka Sasano Yoshiki Nakahara Maki Katsuhara 《Physiologia plantarum》2017,159(1):120-128
A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H2O2 permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H2O2‐transporting aquaporins among 15 barley and rice TIPs. Influx of H2O2 into yeast cells expressing HvPIP2;5 was determined with a florescent‐dye‐based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site‐directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser‐126 in HvPIP2;5 has a large impact on H2O2 transport with a minor influence on the HvPIP2;5‐mediated water transport. 相似文献
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Functional analysis of water channels in barley roots 总被引:1,自引:0,他引:1
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Characterization of CAX4, an Arabidopsis H(+)/cation antiporter 总被引:1,自引:0,他引:1
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Landau BR Spring-Robinson CL Muzic RF Rachdaoui N Rubin D Berridge MS Schumann WC Chandramouli V Kern TS Ismail-Beigi F 《American journal of physiology. Endocrinology and metabolism》2007,293(1):E237-E245
Glucose transport rates are estimated noninvasively in physiological and pathological states by kinetic imaging using PET. The glucose analog most often used is (18)F-labeled 2FDG. Compared with glucose, 2FDG is poorly transported by intestine and kidney. We examined the possible use of 6FDG as a tracer of glucose transport. Lacking a hydroxyl at its 6th position, 6FDG cannot be phosphorylated as 2FDG is. Prior studies have shown that 6FDG competes with glucose for transport in yeast and is actively transported by intestine. Its uptake by muscle has been reported to be unresponsive to insulin, but that study is suspect. We found that insulin stimulated 6FDG uptake 1.6-fold in 3T3-L1 adipocytes and azide stimulated the uptake 3.7-fold in Clone 9 cells. Stimulations of the uptake of 3OMG, commonly used in transport assays, were similar, and the uptakes were inhibited by cyclochalasin B. Glucose transport is by GLUT1 and GLUT4 transporters in 3T3-L1 adipocyte and by the GLUT1 transporter in Clone 9 cells. Cytochalasin B inhibits those transporters. Rats were also imaged in vivo by PET using 6(18)FDG. There was no excretion of (18)F into the urinary bladder unless phlorizin, an inhibitor of active renal transport, was also injected. (18)F activity in brain, liver, and heart over the time of scanning reached a constant level, in keeping with the 6FDG being distributed in body water. In contrast, (18)F from 2(18)FDG was excreted in relatively large amounts into the bladder, and (18)F activity rose with time in heart and brain in accord with accumulation of 2(18)FDG-6-P in those organs. We conclude that 6FDG is actively transported by kidney as well as intestine and is insulin responsive. In trace quantity, it appears to be distributed in body water unchanged. These results provide support for its use as a valid tracer of glucose transport. 相似文献
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Mineo Shibasaka Sizuka Sasano Sigeko Utsugi Maki Katsuhara 《Plant signaling & behavior》2012,7(12):1648-1652
Water homeostasis is crucial to the growth and survival of plants. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. We characterized a novel PIP2 gene, HvPIP2;8 in barley (Hordeum vulgare). HvPIP2;8 shared 72–76% identity with other HvPIP2s and 74% identity with rice OsPIP2;8. The gene was expressed in all organs including the shoots, roots and pistil at a similar level. When HvPIP2;8 was transiently expressed in onion epidermal cells, it was localized to the plasma membrane. HvPIP2;8 showed transport activity for water in Xenopus oocytes, however its interaction with HvPIP1;2 was not observed. These results suggest that HvPIP2;8 plays a role in water homeostasis although further functional analysis is required in future. 相似文献
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Background
The essentiality of boron (B) for plant growth was established >85 years ago. In the last decade, it has been revealed that one of the physiological roles of B is cross-linking the pectic polysaccharide rhamnogalacturonan II in primary cell walls. Borate cross-linking of pectic networks serves both for physical strength of cell walls and for cell adhesion. On the other hand, high concentrations of B are toxic to plant growth. To avoid deficiency and toxicity problems, it is important for plants to maintain their tissue B concentrations within an optimum range by regulating transport processes. Boron transport was long believed to be a passive, unregulated process, but the identification of B transporters has suggested that plants sense and respond to the B conditions and regulate transporters to maintain B homeostasis.Scope
Transporters responsible for efficient B uptake by roots, xylem loading and B distribution among leaves have been described. These transporters are required under B limitation for efficient acquisition and utilization of B. Transporters important for tolerating high B levels in the environment have also been identified, and these transporters export B from roots back to the soil. Two types of transporters are involved in these processes: NIPs (nodulin-26-like intrinsic proteins), boric acid channels, and BORs, B exporters. It is demonstrated that the expression of genes encoding these transporters is finely regulated in response to B availability in the environment to ensure tissue B homeostasis. Furthermore, plants tolerant to stress produced by low B or high B in the environment can be generated through altered expression of these transporters.Conclusions
The identification of the first B transporter led to the discovery that B transport was a process mediated not only by passive diffusion but also by transporters whose activity was regulated in response to B conditions. Now it is evident that plants sense internal and external B conditions and regulate B transport by modulating the expression and/or accumulation of these transporters. Results obtained in model plants are applicable to other plant species, and such knowledge may be useful in designing plants or crops tolerant to soils containing low or high B. 相似文献13.
Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots 总被引:1,自引:0,他引:1
Horie T Kaneko T Sugimoto G Sasano S Panda SK Shibasaka M Katsuhara M 《Plant & cell physiology》2011,52(4):663-675
Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed. 相似文献
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Xue-Ning Zhang Zhen-Hai Han Li-Li Yin Jin Kong Xue-Feng Xu Xin-Zhong Zhang Yi Wang 《Molecular biology reports》2013,40(2):1499-1504
Malus xiaojinensis is an important, iron-efficient rootstock germplasm. Iron uptake is an elaborately controlled process in plant roots, involving specialized transporters. MxIRT1, a Fe(II) transporter gene of M. xiaojinensis, is homologous to other iron transporters at the amino acid level. In the current study, the plasmid pYES2.0-MxIRT1, containing MxIRT1 cDNA, was constructed and transformed into yeast mutants. The results indicated that it could reverse the phenotype of yeast strain DEY1453, an iron uptake mutant. Complementation tests suggested that it might not be a specific transporter, as it was able to restore the phenotypes of other yeast mutant strains, including Mn, Cu and Zn uptake mutants. The functions of the critical histidine residues in the His-box of MxIRT1 were tested by transforming mutant yeast strain DEY1453 with different His residues altered by directed mutagenesis. The His-box of MxIRT1 was found to be necessary for iron transport, with different histidine residues (H1–4) playing different roles in the transport. 相似文献
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Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato 总被引:14,自引:0,他引:14
Bereczky Z Wang HY Schubert V Ganal M Bauer P 《The Journal of biological chemistry》2003,278(27):24697-24704
Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation. 相似文献
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Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi. 相似文献
17.
植物质膜水通道蛋白(plasma membrane intrinsic proteins,PIPs)是位于细胞质膜上具有选择性、高效转运水分的一类膜内在蛋白,参与植物生长发育的多个生理活动。本研究以大麦‘Haruna—nijo’为材料,对水培幼苗进行4℃冷胁迫,采用实时荧光定量PCR技术对胁迫期(4℃,48h)和温度恢复期(16℃,48h)两个过程的水通道蛋白PIPSs基因表达进行了分析;同期测定了根水导度(Lpr)、根长和苗高,分析冷胁迫下大麦根mF基因的表达与水分生理的关系。结果表明:大麦幼苗经4℃低温胁迫48h后,苗的生长明显受抑,根的生长无显著变化;温度恢复48h后,苗恢复生长,根的生长无显著变化;根水导度在胁迫期下降,恢复期急剧升高,均无显著差异。实时荧光定量PCR结果显示,根中表达量最高的是HvPIP1;2和HvPIP1;3,最低的是HvPIP1;1和HvPIP2;3;冷处理后HvPIPs表达童与对照比较总体百降,其HvPIP1;2、HvPIP1;3、HvPIP1;4、HvPIP1;5、HvPIP2;1、HvPIP2;2明显下调。恢复后大多数HvPIPS表达童增加.HvPIP1;1、HvPIP1;2、HvPIP1;5、HvPIP2;3显砉增如,HvPIP1;4、mPIP2;5表达量降低,但无显著轰异,研菀发现,冷弼迫后夫菱粮HvPIPs的表达情况总体下调,恢复生长大部分HvPIPs上调,结合根水导度的变化,推测大麦HvPIPs在抗冷反应中的作用复杂,冷害的不同阶段HvPIPs对水分吸收所起的作用不同。 相似文献
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Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis. The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles. In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes. Each null mutation exhibited sensitivity to metal ion chelators at different concentrations. The combination of null mutants DeltaSMF1 + DeltaSMF2 and the triple null mutant Delta3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively. Addition of 5 microm copper or 25 microm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent. The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein. Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Delta3SMF. Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes. The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3 and FET4 were inactivated. Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium. We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells. 相似文献
19.
Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet 总被引:1,自引:0,他引:1
Yamada N Sakakibara S Tsutsumi K Waditee R Tanaka Y Takabe T 《Journal of plant physiology》2011,168(14):1609-1616
Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells. 相似文献
20.
Ashikov A Routier F Fuhlrott J Helmus Y Wild M Gerardy-Schahn R Bakker H 《The Journal of biological chemistry》2005,280(29):27230-27235
The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus. 相似文献