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The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming a1 subunit and two auxiliary β and α2d subunits. Although auxiliary subunits are critical for channel function, whether they are required for a1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/a1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2d, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes. [ABSTRACT FROM AUTHOR]

The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis. [ABSTRACT FROM AUTHOR]

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2- O-β- d-glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane-enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A1 (vacuolar H+-ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H+-antiporter. Despite its absence in the vacuole, we observed the MgATP-dependent uptake of SGE by Arabidopsis vacuolar membrane-enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A1 and gramicidin D providing evidence that uptake was dependent on an H+-antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP-dependent SAG and SGE uptake exhibited Michaelis-Menten-type saturation kinetics. The vacuolar enriched-membrane vesicles had a 46-fold greater affinity and a 10-fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long-term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations. [ABSTRACT FROM AUTHOR]

Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H+ symporter in an inward-facing conformation at 3.2-Å resolution. The Staphylococcus epidermidis glucose/H+ symporter is homologous to human glucose transporters, is very specific and has high avidity for glucose, and is inhibited by the human glucose transport inhibitors cytochalasin B, phloretin, and forskolin. On the basis of the crystal structure in conjunction with mutagenesis and functional studies, we propose a mechanism for glucose/H+ symport and discuss the symport mechanism versus facilitated diffusion. [ABSTRACT FROM AUTHOR]

According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H+ symport, a cavity facing the periplasmic side is thought to open with closure of the inward-facing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electron-electron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxide-labeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-154→Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 Å. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 Å. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-154→Gly mutant, sugar-induced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY. [ABSTRACT FROM AUTHOR]

Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L. mesenteroides contain minute amounts of enolase, which separate as small crystals. Expression of L. mesenteroides enolase in Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing the conversion of 2-phsopho-d-glycerate to phosphoenolpyruvate. The pH optimum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s−1, respectively. At phosphate concentrations of 1 mM and below, fluoride is a noncompetitive inhibitor with respect to 2-phospho-d-glycerate, but in the presence of 20 mM phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase significantly inhibits the activity of purified dextransucrase, and does not bind human plasminogen. Results here suggest that in some organisms enolase may participate in protein interactions that have no direct relevance to pathogenic invasion. [ABSTRACT FROM AUTHOR]