Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys(54) in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys(54) α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit.

Regulated exocytosis of an H+/myo-inositol symporter at synapses and growth cones.

Phosphoinositides, synthesized from myo-inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H(+)/myo-inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense-core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo-inositol uptake. The symporter is then endocytosed in a dynamin-dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity-dependent regulation of myo-inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.

A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability.

Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated.

Mobile gibberellin directly stimulates Arabidopsis hypocotyl xylem expansion.

Secondary growth of the vasculature results in the thickening of plant structures and continuously produces xylem tissue, the major biological carbon sink. Little is known about the developmental control of this quantitative trait, which displays two distinct phases in Arabidopsis thaliana hypocotyls. The later phase of accelerated xylem expansion resembles the secondary growth of trees and is triggered upon flowering by an unknown, shoot-derived signal. We found that flowering-dependent hypocotyl xylem expansion is a general feature of herbaceous plants with a rosette growth habit. Flowering induction is sufficient to trigger xylem expansion in Arabidopsis. By contrast, neither flower formation nor elongation of the main inflorescence is required. Xylem expansion also does not depend on any particular flowering time pathway or absolute age. Through analyses of natural genetic variation, we found that ERECTA acts locally to restrict xylem expansion downstream of the gibberellin (GA) pathway. Investigations of mutant and transgenic plants indicate that GA and its signaling pathway are both necessary and sufficient to directly trigger enhanced xylogenesis. Impaired GA signaling did not affect xylem expansion systemically, suggesting that it acts downstream of the mobile cue. By contrast, the GA effect was graft transmissible, suggesting that GA itself is the mobile shoot-derived signal.

Deubiquitylation regulates activation and proteolytic cleavage of ENaC

The epithelial sodium channel (ENaC) is critical for sodium and BP homeostasis. ENaC is regulated by Nedd4-2-mediated ubiquitylation, which leads to its internalization; this process can be reversed by deubiquitylation, which is regulated by the aldosterone-induced enzyme Usp2-45. In a second regulatory pathway, ENaC can be activated by luminal serine protease-mediated cleavage of its extracellular loops. Whether these two regulatory processes interact, however, is unknown. Here, in HEK293 cells stably transfected with ENaC, Usp2-45 interacted with ENaC, leading to deubiquitylation of the channel and stimulation of ENaC activity >20-fold. This was accompanied by a modest increase in cell surface expression of ENaC and by proteolytic cleavage of alphaENaC and gammaENaC at their extracellular loops. When endocytosis was inhibited with dominant negative dynamin (DynK44R), channel density and gammaENaC cleavage were increased, but alphaENaC cleavage and ENaC activity were not augmented. When Usp2-45 was coexpressed with DynK44R, both alphaENaC cleavage and activity were recovered. In summary, these data suggest that Usp2-45 deubiquitylation of ENaC enhances the proteolytic activation of both alphaENaC and gammaENaC, possibly by inducing a conformational change and by interfering with endocytosis, respectively

Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities.

Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA-gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.

Notch1 expression on T cells is not required for CD4+ T helper differentiation.

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.

Shedding light on proteolytic cleavage of CD44: the responsible sheddase and functional significance of shedding.

CD44 is the major cell-surface receptor for hyaluronan, which is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. Studies have shown that CD44-dependent migration requires CD44 to be shed from the cell surface and that matrix metalloproteinase-mediated cleavage may provide an underlying mechanism. However, the full spectrum of proteases that may participate in CD44 shedding has yet to be defined. In this issue, Anderegg et al. demonstrate that ADAM10, but not ADAM17 or MMP14, mediates constitutive shedding of CD44 in human melanoma cells and that knockdown of ADAM10 blocks the antiproliferative activity of the soluble proteolytic cleavage product of CD44.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Knock, knock, knocking on muscle doors. Visions on the transport of substrates across the plasma membrane in muscle.

Muscle is a major player in metabolism. It uses large amounts of glucose in the absorptive state and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. Lipid substrates such as fatty acids or ketone bodies are preferentially used by muscle in certain physiological conditions. Muscle is also the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters such as glucose carriers, carnitine, creatine or amino acid transporters maintain muscle metabolism by taking up or releasing substrates or metabolites across the cell surface. The goal of this review is the molecular characterization of muscle membrane transporter proteins and the analysis of their regulatory roles.

Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection.

α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.