Requirements for catalysis in mammalian glycogenin

Glycogenin is a glycosyltransferase that functions as the autocatalytic initiator for the synthesis of glycogen in eukaryotic organisms. Prior structural work identified the determinants responsible for the recognition and binding of UDP-glucose and the catalytic manganese ion and implicated two aspartic acid residues in the reaction mechanism for self-glucosylation. We examined the effects of substituting asparagine and serine for the aspartic acid residues at positions 159 and 162. We also examined whether the truncation of the protein at residue 270 (delta270) was compatible with its structural integrity and its functional role as the initiator for glycogen synthesis. The truncated form of the enzyme was indistinguishable from the wild-type enzyme by all measures of activity and could support glycogen accumulation in a glycogenin-deficient yeast strain. Substitution of aspartate 159 by either serine or asparagine eliminated self-glucosylation and reduced trans-glucosylation activity by at least 260-fold but only reduced UDP-glucose hydrolytic activity by 4-14-fold. Substitution of aspartate 162 by either serine or asparagine eliminated self-glucosylation activity and reduced UDP-glucose hydrolytic activity by at least 190-fold. The trans-glucosylation of maltose was reduced to undetectable levels in the asparagine 162 mutant, whereas the serine 162 enzyme showed only an 18-30-fold reduction in its ability to trans-glucosylate maltose. These data support a role for aspartate 162 in the chemical step for the glucosyltransferase reaction and a role for aspartate 159 in binding and activating the acceptor molecule.     

Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.     

Endocytosis: driving membranes around the bend

When a nascent vesicle buds, the membrane must curve. Several mechanisms have been proposed for curvature creation or stabilization. Structural analysis of the ENTH domain of the endocytic protein epsin has suggested a new mechanism, in which the ENTH domain pushes its way into membranes, thus bending them into shape.     

Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.     

Effects of phthalate esters on actin cytoskeleton of Py1a rat osteoblasts

We evaluated, by confocal laser scanning microscopy, the actin cytoskeleton of immortalized rat Py1a osteoblasts treated with phthalate esters (butyl benzyl phthalate, BBP and dibutyl phthalate, DBP), endocrine disruptors with estrogenic activity. We observed some peculiar modifications of actin cytoskeleton and cells changing from a spindle shape to a rounded form. In particular, F-actin formed thick bundles around the cell membrane but only a weak labeling was observed in rounded cells. Also influence on apoptosis and short-term effects on FGF-2 were studied. It was found that BBP and DBP exert their action in a similar way, act in a transient manner and do not induce apoptosis.     

The cellulose-deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II,an enzyme processing N-glycans during ER quality control

rsw3 is a temperature-sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map-based strategy, and shows high similarity to the catalytic alpha-subunits of glucosidase II from mouse, yeast and potato. These enzymes process N-linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative beta-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with beta-subunits in mammals and yeast. The genes encoding the putative alpha- and beta-subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose-deficient mutants, but some--notably, production of multiple rosettes and a lack of secreted seed mucilage--are not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.     

Mechanism of ubiquitin recognition by the CUE domain of Vps9p

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.     

Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.