Expression of renal organic cation transporter in Xenopus laevis oocytes.
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ABSTRACT:
SUBMITTER: Hori R
PROVIDER: S-EPMC1131049 | biostudies-other | 1992 Apr
REPOSITORIES: biostudies-other
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Project description:BackgroundRapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.ResultsWe used the generally available software (http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/) to predict a DNA sequence for the plant gene that is better suited for Xenopus laevis. Rice OsNAR2.1 and OsNRT2.3a DNA optimized sequences were commercially synthesized for Xenopus expression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and original OsNAR2.1 and OsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3- than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.ConclusionThe functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in the Xenopus oocyte system.
| S-EPMC3878178 | biostudies-literature
Project description:The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.
| S-EPMC4186817 | biostudies-literature
Project description:Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes.
| S-EPMC1151387 | biostudies-other
Project description:Microinjection of mRNA isolated from rat kidney cortex into Xenopus laevis oocytes resulted in the expression of a Na(+)-dependent dicarboxylate transporter, as detected by uptake measurements with [14C]succinate as substrate. The expressed transporter showed an S-shaped Na(+)-dependence with half-maximal activation at 19-21 mM Na+ and a Hill coefficient between 2 and 3. Endogenous succinate uptake was not Na(+)-dependent. Na(+)-stimulated succinate uptake in mRNA-injected oocytes exhibited a maximum at pH 7.5, whereas endogenous Na(+)-independent transporter was fastest at pH 8.5. The expressed dicarboxylate transporter also differed from the endogenous transporter in its sensitivity to citrate as well as dicarboxylates in trans and cis configurations. The expressed transporter resembled the renal basolateral transporter, especially with respect to affinity for succinate (Km 28 microM), activation by Na+, pH-dependence and substrate specificity. After injection of size-fractionated mRNA, succinate uptake was expressed by mRNA of 2-3 kb. Our results suggest expression of the basolateral Na(+)-dependent dicarboxylate transporter after injection of mRNA from rat kidney into Xenopus oocytes.
| S-EPMC1137786 | biostudies-other
Project description:Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevis oocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H(+) gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a K(m) value of 0.74+/-0.07 mM was determined at pH 7.0. For the other substrates, K(i) values between 100 microM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by alpha-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.
| S-EPMC1220388 | biostudies-other
Project description:MicroRNA-protein complexes (microRNPs) can activate translation of target reporters and specific mRNAs in quiescent (i.e., G0) mammalian cell lines. Induced quiescent cells, like folliculated immature oocytes, have high levels of cAMP that activate protein kinase AII (PKAII) to maintain G0 and immature states. We report microRNA-mediated up-regulated expression of reporters in immature Xenopus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that maintenance of cAMP levels and downstream PKAII signaling are required for microRNA-mediated up-regulated expression in oocytes. We identify an important, endogenous cell state regulator, Myt1 kinase, as a natural target of microRNA-mediated up-regulation in response to xlmiR16, ensuring maintenance of oocyte immaturity. Our data reveal the physiological relevance of cAMP/PKAII-controlled posttranscriptional gene expression activation by microRNAs in maintenance of the immature oocyte state.
| S-EPMC3100953 | biostudies-literature
Project description:We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca2+ release comes from a region of width approximately 450 nm, that the release duration is peaked around 18 s and that the underlying Ca2+ currents range between 0.12 and 0.95 pA. All these parameters are independent of IP(3) concentration. We explore what distributions of channels that open during a puff, N(p), and what relations between current and number of open channels, I(N(p)), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al. To this end, we use simple "mean field" models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(N(p)) approximately N(p) for small N(p) and I(N(p)) approximately N(p)(1/alpha) (alpha > 1) for large N(p), provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of approximately 400 puffs obtained using the dye Oregon green.
| S-EPMC2839043 | biostudies-literature
Project description:The nucleolus is a multi-compartment, non-membrane-bound organelle within the nucleus. Nucleolar assembly is influenced by proteins capable of phase separation. Xenopus laevis oocytes contain hundreds of large nucleoli that provide experimental access for nucleoli that is unavailable in other systems. Here we detail methods to streamline the in vivo analysis of the compartmentalization of nucleolar proteins that are suspected of phase separation. The nucleolus is the main hub of ribosome biogenesis and here we present data supporting the division of proteins into nucleolar domains based on their function in ribosome biogenesis. We also describe the use of vital dyes such as Hoechst 33342 and Thioflavin T in nucleolar staining. Additionally, we quantify nucleolar morphology changes induced by heat shock and actinomycin D treatments. We suggest these approaches will be valuable in a variety of studies that seek to better understand the nucleolus, particularly those regarding phase separation. These approaches may also be instructive for other studies on phase separation, especially in the nucleus.
| S-EPMC9474603 | biostudies-literature
Project description:RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.
| S-EPMC3549563 | biostudies-literature
Project description:Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.
| S-EPMC1149627 | biostudies-other