Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K-0.5 = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1 ',6 '-trichloro-4,1 ',6 '-trideoxygalactosucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K-i) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, Delta S1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical Delta S1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, mu 1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.

Flagellin of Pseudomonas aeruginosa inhibits Na+ transport in airway epithelia

Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

Anti-proliferative effects of novel Glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)2 group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 μM and 10 μM compound (11) and 10 μM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/ or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness. © 2006 Bentham Science Publishers Ltd.