Electrical potential differences across salt transporting membranes.

Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1.

Biological Transport↗

PubMed2003

Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane.

In higher plants, two independent pathways are responsible for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central five-carbon precursors of all isoprenoids. The cytosolic pathway, which involves mevalonate (MVA) as a key intermediate, provides the precursor molecules for sterols, ubiquinone, and certain sesquiterpenes, whereas the plastidial MVA-independent pathway is involved in the formation of precursors for the biosynthesis of isoprene, monoterpenes, diterpenes, carotenoids, abscisic acid, and the side chains of chlorophylls, tocopherols, and plastoquinone. Recent experiments provided indirect evidence for the presence of an export system for isoprenoid intermediates from the plastids to the cytosol in Arabidopsis thaliana. Here we report that isolated chloroplasts (from spinach, kale, and Indian mustard), envelope membrane vesicles, and proteoliposomes prepared from the solubilized proteins of envelope membranes (from spinach) are capable of the efficient transport of isopentenyl diphosphate and geranyl diphosphate. Lower rates of transport were observed with the substrates farnesyl diphosphate and dimethylallyl diphosphate, whereas geranylgeranyl diphosphate and mevalonate were not transported with appreciable efficiency. Our data suggest that plastid membranes possess a unidirectional proton symport system for the export of specific isoprenoid intermediates involved in the metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis.

Biological Transport↗

PubMed2003

Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two systems have been shown to be involved in the active transport of inorganic phosphate (P(i)) across the plasma membrane, the high-affinity system and the low-affinity system. The high-affinity system consists of Pho84p and Pho89p. The low-affinity system has recently been shown to be composed of Pho87p, Pho90p, and Pho91p. In this study, we found that the Deltapho87Deltapho90Deltapho91 strain which shows repressed PHO5 expression under high-P(i) condition has, unlike the wild-type strain, increased levels of PHO5 expression at an intermediate P(i) concentration of 0.5mM, whereas it is not defective in terms of P(i) uptake under the same conditions. Moreover, we observed that the transcription levels of PHO84 and PHO89 are also increased in low-affinity P(i)-transporter-defective mutants, indicating that the inactivation of low-affinity P(i) transporters leads to the activation of the PHO pathway. In contrast to that of PHO5, PHO84, and PHO89, the transcription of PHO87, PHO90, and PHO91 genes is independent of P(i) concentration and Pho4p activity, and the increased expression level of these transporters does not occur when other transporters including PHO84 are inactivated. The fact that low-affinity P(i)-transporter-defective mutants exhibit a derepression of P(i)-responsive genes suggests that low-affinity transporters play a role not only in P(i) transport but also in the regulation of the P(i) signal transduction pathway.

Biological Transport↗