Nitrate is both a nitrogen source for higher plants and a signal molecule regulating their development. In Arabidopsis, the NRT1.1 nitrate transporter is crucial for nitrate signaling governing root growth, and has been proposed to act as a nitrate sensor. However, the sensing mechanism is unknown. Herein we show that NRT1.1 not only transports nitrate but also facilitates uptake of the phytohormone auxin. Moreover, nitrate inhibits NRT1.1-dependent auxin uptake, suggesting that transduction of nitrate signal by NRT1.1 is associated with a modification of auxin transport. Among other effects, auxin stimulates lateral root development. Mutation of NRT1.1 enhances both auxin accumulation in lateral roots and growth of these roots at low, but not high, nitrate concentration. Thus, we propose that NRT1.1 represses lateral root growth at low nitrate availability by promoting basipetal auxin transport out of these roots. This defines a mechanism connecting nutrient and hormone signaling during organ development.

Different distribution patterns of the arginine/H؉ symporter Can1p, the H ؉ plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP-and Pma1p-RFP-containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell. INTRODUCTIONLipid rafts (detergent-resistant membranes, detergent-insoluble glycolipid-enriched microdomains, or glycolipid-enriched membranes) are dynamic assemblies enriched in sterols and sphingolipids occurring laterally distributed in the plasma membrane of most, if not all, eukaryotes (for reviews, see Simons and Ikonen, 1997;Brown and London, 1998). In mammalian cells cholesterol, sphingomyelin, and glycosphingolipids are the basic constituents of detergentresistant membranes (Rietveld and Simons, 1998), whereas in yeast these are ergosterol, inositolphosphoceramide, and its mannosylated derivatives (Kü bler et al., 1996;Bagnat et al., 2000). Existence of similar plasma membrane domains in plant cells can also be expected, although it has not been reported so far.A distinctive feature of lipid rafts is their insolubility in mild nonionic detergents (typically Triton X-100) at 4°C (Brown and Rose, 1992;Rietveld and Simons, 1998). Consequently, they are found floating in low-density fractions of solubilizates of mild detergent-treated membranes. Rafts obtained by this procedure selectively recruit specific membrane proteins, whereas others are excluded. The recognition of selective protein retention within the detergent-resistant domains led to a concept of lateral subcompartmentation within the plasma membrane (Simons and Ikonen, 1997;Brown and London, 1998).It was proposed that rafts form a platform for lipid (Simons and Ikonen, 2000) and protein sorting and trafficking (Simons and van Meer, 1988;Galbiati et al., 2001;Ikonen, 2001) and cell signaling (Field et al., 1997;Stauffer and Meyer, 1997;Simons and Toomre, 2000;Dykstra et al., 2001). Lipid rafts provide the cells also with a mechanism for functional and spatial control of exocytosis (Chamberlain et al., 2001) and are involved in immune cell activation (for reviews, see Dykstra et al., 2001;Galbiati et al., 2001;Katagiri et al., 2001). Accumulating evidence documents that compositionally distinct lipid microdomains are assembled ...

Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2α/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Δlsm4ΔC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2α became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2α. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

Recently, lipid-raft-based subdomains within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using green fluorescent protein fusions, and non-overlapping subdomains containing either Pma1p or Can1p were distinguished. In this study, the long-term stability of the subdomains was investigated. Experiments with latrunculin A and nocodazole ruled out the involvement of cytoskeletal components in the stabilization of the subdomains. Also a putative role of the cell wall was excluded, because protoplasting of the cells changed neither the pattern nor the stability of the subdomains. By contrast, the expected inner dynamics of the membrane subdomains was documented by FRAP experiments. Finally, two other proteins were localized within the frame of the Can1p/Pma1p plasma-membrane partition. We show that Fur4p (another H+ symporter) and Sur7p (a protein of unknown function) occupy the Can1p subdomain.

Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.