Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. (65/7411)

Binding of 14-3-3 proteins near the nuclear localization sequence of Xenopus Cdc25 suppresses its ability to induce entry into mitosis. We have examined the intracellular localization of green fluorescent protein (GFP)-tagged wild-type Cdc25 or a mutant (S287A) that cannot bind 14-3-3 proteins. Upon coexpression with Myc-14-3-3epsilon, GFP-Cdc25-WT was predominantly cytoplasmic, whereas GFP-Cdc25-S287A was exclusively nuclear. Leptomycin B, an inhibitor of nuclear export, elicited a prompt redistribution of GFP-Cdc25-WT to the nucleus. Mutagenesis experiments demonstrated that Cdc25 contains multiple nuclear export sequences. These studies indicate that the binding of 14-3-3 proteins and nuclear export regulate the intracellular localization of Cdc25.  (+info)

The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. (66/7411)

The constitutive transport element (CTE) encoded by simian type D retroviruses directs unspliced viral RNAs into a nuclear export pathway that is congruent with the pathway used by cellular mRNAs. Here, we show that quail cells are refractory to CTE function but become highly permissive upon expression of the human Tap protein, a candidate CTE cofactor. Tap contains a novel sequence-specific RNA binding domain that is sufficient for CTE binding but inadequate to support CTE function. Using microinjection assays, we have defined two NLSs and one NES in Tap. Mutational inactivation of the Tap NES, which lies outside the RNA-binding domain, not only blocks Tap function but also generates dominant-negative forms of Tap. Whereas replacement of the Tap NES with the well-defined Rev NES rescues the ability of Tap to support CTE function, this substitution also confers sensitivity to agents that block the activity of Crm1, the Rev NES cofactor. Together, these data validate Tap as the first human sequence-specific nuclear mRNA export factor and identify a novel type of NES that can support nuclear mRNA export but does not act via Crm1.  (+info)

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization. (67/7411)

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.  (+info)

Primary active transport of organic anions on bile canalicular membrane in humans. (68/7411)

Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2, 4-Dinitrophenyl-S-glutathione (DNP-SG), leukotriene C4 (LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), beta-estradiol 17-(beta-D-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2-17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was approximately 3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.  (+info)

The uptake and metabolism of uridine by the slime mould Physarum polycephalum. (69/7411)

1. Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29 muM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine. 2. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides. 3. As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C-2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.  (+info)

Passive potassium transport in LK sheep red cells. Effects of anti-L antibody and intracellular potassium. (70/7411)

The passive K influx in low K(LK) red blood cells of sheep saturates with increasing external K concentration, indicating that this mode of transport is mediated by membrane-associated sites. The passive K influx, iMLK, is inhibited by external Na. Isoimmune anti-L serum, known to stimulate active K transport in LK sheep red cells, inhibits iMLK about twofold. iMLK is affected by changes in intracellular K concentration, [K]i, in a complex fashion: increasing [K]i from near zero stimulates iMLK, while further increases in [K]i, above 3 mmol/liter cells, inhibit iMLK. The passive K influx is not mediated by K-K exchange diffusion. The effects of anti-L antibody and [K]i on passive cation transport are specific for K: neither factor affects passive Na transport. The common characteristics of passive and active K influx suggest that iMLK is mediated by inactive Na-K pump sites, and that the inability to translocate Na characterizes the inactive pumps. Anti-L antibody stimulates the K pump in reticulocytes of LK sheep. However, anti-L has no effect on iMLK in these cells, apparently because reticulocytes do not have the inactive pump sites which, in mature LK cells, are a consequence of the process of maturation of circulating LK cells. The results also indicate that anti-L alters the maximum velocity of both active and passive K fluxes by converting pumps sites from a form mediating passive K influx to an actively transporting form.  (+info)

Response to hypertonicity in mesothelial cells: role of Na+/myo-inositol co-transporter. (71/7411)

BACKGROUND: During peritoneal dialysis, the peritoneal mesothelium is exposed continually to hypertonic dialysates. The purpose of this study is to see if rat mesothelial cells have an osmoregulatory mechanism to adapt to hypertonic environment. METHODS: The intracellular content of organic osmolytes was measured by HPLC methods. Myo-inositol transport activity was measured by Na+-dependent uptake of [3H]myo-inositol. mRNA abundance for the Na+/myo-inositol co-transporter (SMIT) was examined by Northern and slot-blot analyses. RESULTS: In isotonic mesothelial cells, only myo-inositol could be detected. After switching to hypertonic medium made by addition of NaCl, myo-inositol content gradually increased and peaked at 48 h after the switch. The myo-inositol content in hypertonic cells increased > 7-fold over the value in isotonic cells. The contents of betaine and glycerophosphorylcholine (GPC) also increased but were less than that of myo-inositol. Sorbitol was not accumulated in this condition. When glucose was used to increase medium osmolality, all of the four osmolytes were increased by hypertonicity (myo-inositol > sorbitol > GPC > betaine). Thus, myo-inositol is the most abundant osmolyte in the mesothelial cells. Na+-dependent myo-inositol uptake in hypertonic cells was approximately 7-fold the uptake in isotonic cells, reaching a maximum 16 h after switching to a hypertonic medium. The uptake rate increased as medium osmolality increased from 300 to 500 mosm/kg. SMIT mRNA rapidly increased after increasing medium osmolality, reaching a maximum 8 h after the switch. The relative increase in the mRNA abundance was approximately 11 times isotonic levels. CONCLUSIONS: Mesothelial cells respond to extracellular hypertonicity by increasing SMIT mRNA abundance, myo-inositol transport activity and accumulating myo-inositol into the cells.  (+info)

Interference of nucleoside diphosphate derivatives of 2-deoxy-D-glucose with the glycosylation of virus-specific glycoproteins in vivo. (72/7411)

The predominant effect of 2-deoxy-D-glucose on chick embryo cells infected with Semliki Forest virus is an interference with glycosylation of virus-specific glycoproteins; this results in a block of synthesis of infectious virus. Incorporation of radioactive mannose is blocked severely in the presence of 2-deoxyglucose in the cultural medium although it is readily phosphorylated and subsequently activated by GTP to yield GDP-mannose, which accumulates under these conditions. The intracellular concentrations of GDP-mannose and UDP-N-acetyl-D-hexosamine are not reduced in the presence of the inhibitor. An equimolar concentration of mannose in the cultural medium competes with the inhibitory effect of the deoxysugar and drops the cellular pool of GDP-2-deoxy-D-glucose below the level of detection, at the same time restoring the synthesis of infectious virus. When the intracellular concentration of UDP-2-deoxyglucose is reduced by addition of glucose into the cultural medium the inhibition of virus synthesis by the deoxysugar and the concentration of GDP-2-deoxyglucose within the cells remain near to the values when the inhibitor is present alone. It is concluded that among the metabolites of 2-deoxyglucose which occur in vivo after addition of 2-deoxyglucose to the culture medium, GDP-2-deoxyglucose is the agent responsible for inhibition of glycosylation of viral glycoproteins.  (+info)