KEG1/YFR042w encodes a novel Kre6-binding endoplasmic reticulum membrane protein responsible for beta-1,6-glucan synthesis in Saccharomyces cerevisiae.

KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent protein- or Myc(6)-tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in beta-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with Deltakre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of beta-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in beta-1,6-glucan synthesis in S. cerevisiae.

Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis-(2-ethylhexyl)phthalate.

The taxonomic position of an actinomycete strain isolated from soil from Natore, Bangladesh, was examined by using a polyphasic approach. The strain, designated AAB-4(T), was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded spores. The 16S rRNA gene of strain AAB-4(T) was sequenced directly and then compared with those of previously studied streptomycetes following the generation of two phylogenetic trees by using maximum-likelihood and neighbour-joining algorithms. This confirmed the assignment of the novel strain to the genus Streptomyces. This strain showed a high level of 16S rRNA gene sequence similarity to Streptomyces thermoviolaceus, Streptomyces thermodiastaticus and Streptomyces longisporus, among others, but could be distinguished from them by phenotypic and physiological traits. This micro-organism produces bis-(2-ethylhexyl)phthalate, an antibacterial and antifungal agent. It is proposed that strain AAB-4(T) be classified as a novel species within the genus Streptomyces, as Streptomyces bangladeshensis sp. nov. (type strain, AAB-4(T)=LMG 22738(T)=NRRL B-24326(T)).

Heterologous expression and characterization of Schizosaccharomyces pombe vacuolar carboxypeptidase Y in Saccharomyces cerevisiae.

To investigate the intracellular transport mechanism of the vacuolar carboxypeptidase of Schizosaccharomyces pombe (SpCPY), SpCPY was expressed in Saccharomyces cerevisiae and its biosynthesis and sorting were examined. When Sac. cerevisiae prc1Delta, devoid of intrinsic (Sc) CPY activity, was transformed with a plasmid carrying the Sch. pombe cpy1(+) gene, CPY activity was restored. Pulse-chase experiments revealed that SpCPY is initially synthesized in a pro-precursor form and then converted to a heterodimer, the mature form, in Sac. cerevisiae cells. SpCPY was not processed into intermediate or mature forms in pep4 mutant cells, indicating that SpCPY was proteolytically cleaved in a PEP4-dependent manner in Sac. cerevisiae. Several vps mutants, which are defective in vacuolar protein-sorting, exhibited a defect in the maturation of SpCPY. Moreover, the maturation of SpCPY was severely inhibited in a vps10 strain, although the pro- segment of SpCPY does not contain a QRPL-like sequence, which is the putative targeting signal of ScCPY. When SpCPY was expressed in a wild-type strain, more than 90% of ScCPY was normally sorted to the vacuole, indicating that SpCPY does not compete with ScCPY for vacuolar sorting. In contrast, expression of SpCPY resulted in a missorting of a ScCPY-invertase fusion protein to the cell surface. These results suggested that there are two different binding sites for SpCPY and ScCPY on Vps10p and that the binding of SpCPY to Vps10p interferes with the binding of a ScCPY-invertase fusion protein.