The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.

Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (Fru-1,6-Pase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) requires two divalent metal ions to hydrolyze alpha-D-fructose 1,6-bisphosphate. Although not required for catalysis, monovalent cations modify the enzyme activity; K+ and Tl+ ions are activators, whereas Li+ ions are inhibitors. Their mechanisms of action are still unknown. We report here crystallographic structures of pig kidney Fru-1,6-Pase complexed with K+, Tl+, or both Tl+ and Li+. In the T form Fru-1,6-Pase complexed with the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P2) and Tl+ or K+ ions, three Tl+ or K+ binding sites are found. Site 1 is defined by Glu-97, Asp-118, Asp-121, Glu-280, and a 1-phosphate oxygen of AhG-1,6-P2; site 2 is defined by Glu-97, Glu-98, Asp-118, and Leu-120. Finally, site 3 is defined by Arg-276, Glu-280, and the 1-phosphate group of AhG-1,6-P2. The Tl+ or K+ ions at sites 1 and 2 are very close to the positions previously identified for the divalent metal ions. Site 3 is specific to K+ or Tl+. In the divalent metal ion complexes, site 3 is occupied by the guanidinium group of Arg-276. These observations suggest that Tl+ or K+ ions can substitute for Arg-276 in the active site and polarize the 1-phosphate group, thus facilitating nucleophilic attack on the phosphorus center. In the T form complexed with both Tl+ and Li+ ions, Li+ replaces Tl+ at metal site 1. Inhibition by lithium very likely occurs as it binds to this site, thus retarding turnover or phosphate release. The present study provides a structural basis for a similar mechanism of inhibition for inositol monophosphatase, one of the potential targets of lithium ions in the treatment of manic depression.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.