The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.

The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apoMtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.In eukaryotic cells, the nuclear and cytoplasmic compartmentalization requires that a large number of molecules be continuously transported through the nuclear pore complex (NPC), 1 a huge macromolecular structure that spans the nuclear envelope. Nucleoporins represent a subset of the NPC components and form a dense network of proteins that line the channel of the NPC. Transport through the channel requires binding of protein or RNA cargoes to soluble transport receptors and is mediated by the phenylalanine-glycine (FG) repeats that characterize most nucleoporins. The two most common repeats that are found in nucleoporins and are often present in many copies along the whole molecule are based on GLFG or FXFG cores (1-3).The best characterized pathway of protein import and export involves members of the conserved family of transport receptors called karyopherins/importin-␤, also known as exportins/ importins. The karyopherin transport factors share a common structural framework and respond to the small GTPase, Ran, to bind or release their cargo within the appropriate cellular compartments. Meanwhile, Ran must also shuttle across the NPC to equilibrate its nuclear level; this is performed via a specific nuclear import factor known as nuclear transport factor 2 (NTF2) (4 -8).Unlike this well established protein transport pathway, no karyopherin family member that would function in general mRNA export has been identified. Instead, recent studies in yeast and metazoans have pointed to several highly conserved proteins that are known as nuclear export factor (NXF) and are specifically required for mRNA export, but do not include karyopherin or NTF2 (9). Among members of the NXF family, the best characterized candidate is the Saccharomyces cerevisiae protein Mex67, whose conserved metazoan counterpart is known as TAP or NXF. Mex67 interacts with both bulk poly(Aϩ) RNA and nuclear p...

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin~AzMC! has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase~SULT1A1 or P-PST-1!. For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein~MBP! and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1~K i ϭ 0.47 6 0.05 mM!. Moreover, enzyme activity toward 2-naphthol was inactivated in a timeand concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.

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