We have sequenced the genetically linked genes for repressible (PHO5) and and constitutive (PHO3) acid phosphatase from S. cerevisiae. Both genes are located on a 3.91 Kb BamHI and HpaI fragment, in the order (5') PHO5, PHO3 (3'). The mRNA transcripts have been analysed by S1-nuclease mapping. They show heterogenous initiation sites. Each of the PHO5 and PHO3 genes codes for 467 amino acids as deduced from the DNA sequence. The coding regions of the two genes show homology both at the nucleotide (82%) and the amino acid (87%) level. In the coding sequences, long stretches of homologous regions are flanked by small non-homologous regions. The nucleotide homology (65%) extends to some length into the 5' and 3' non-coding flanking sequences. Further upstream sequences are unrelated. The comparison of the NH2-terminal amino acid sequence deduced from the nucleotide sequence, with that of purified repressible acid phosphatase revealed the presence of a putative signal peptide.

We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellularly mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.

Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic-and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane-bound 54-kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72-kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54-kDa and 72-kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54-kDa acid phosphatase is glycosylated to the 72-kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72-kDa form. In cells grown in the minimal medium, however, all of the 54-kDa and 72-kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72-kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.Eucaryotic secretory proteins including those of Saccharomyces cerevisiae are synthesized at the endoplasmic reticulum, are transported to the Golgi apparatus and via vesicles to the cell surface. Many of them are N-glycosylated on their secretory pathway. In yeast the pathway of Asn-linked glycosylation starts with the transfer of the oligosaccharide precursor, (GlcNAc)2(Man)9(Glc)3, from the dolichyl diphosphate to appropriate asparagine residues [l, 21. This oligosaccharide is further processed by removal of the three glucose units and a mannose unit [l, 31. Glycoproteins that are secreted have large polymannose chains added to the oligosaccharide core units, whereas glycoproteins destined to remain in the cell have the core oligosaccharide increased by 1 -4 mannose residues [4 -61.Mainly by the use of temperature-sensitive mutants that are blocked at distinct steps in the export of cell-surface glycoproteins, it has been shown that the transfer of the (Glc)3(Man)9(GlcNAc)z core oligosaccharides to the protein occurs in the endoplasmic reticulum and that extension through the addition of outer polymannose chains takes place in Golgi-like organelles [7]. Little is known concerning the regulation of the Asn-linked glycosylation pathway in yeast. Data of Chu and Malley indicate that glucose impairs Nglycosylation of invertase and that the nonglycosylated polypeptide is rapidly degraded by an endogenous protease PI.Correspondence to M. E. Schweingruber, Institut fur Allgemeine Mikrobiologie der Universitat Bern, Baltzerstrasse 4, CH-3012 Bern, SwitzerlandAbbreviations. Endoglycosidase H, endo-N-acetylglucosaminidase H ; Tris/NaCl, 20 mM Tris/HCl, 150 mM NaC1, pH ...

Fission yeast, Schizosaccharomyces pombe, is a natural inositol auxotroph. We show here that the amount of exogenous inositol added to the medium is critical for the control of its life cycle. Above growth-limiting concentrations inositol stimulates mating and sporulation in minimal medium. The effect of inositol is also observed on yeast-extract-medium plates. We selected a mutant, IM49, which mates and sporulates only poorly and show that it is defective in inositol transport. Its defect is in a gene (itr2) coding for a putative 12 membrane-spanning protein. The polypeptide contains the two sugar-transport motifs typical for hexose transporters and shows good homology to the two Saccharomyces cerevisiae inositol transporters. The itr2 gene is essential for cell growth and its mRNA level is repressed by glucose. Mutant IM49 is also complemented by a multicopy suppressor gene (itr1) which codes for a putative hexose transporter with unknown substrate specifity.