Spontaneous recessive orthovanadateresistant mutants of Saccharomyces cerevisiae were obtained in five complementation groups, and all show defects in protein glycosylation that mimic the previously isolated mnn mutants. Three of the groups are allelic to the known mnn8, mnn9, and manlO mutants, whereas the other two groups show other glycosylation defects. The vanadate-resistant phenotype was associated with enhanced hygromycin B sensitivity. The glycosylation phenotypes of the mutants are all reflections of defects in glycoprotein trafficking, and the easy isolation of vanadate-resistant or hygromycin B-sensitive mutants should facilitate the study of this process.Orthovanadate-resistant mutants of Saccharomyces cerevisiae have been studied extensively by Willsky and coworkers (1-3), who identified at least five complementation groups with this phenotype. Considerable effort has been expended to define the site(s) of vanadate action in the sensitive cell and, thereby, provide a clue to the possible mechanism(s) by which vanadate resistance could arise (2, 4). Although a rather high concentration of vanadate (5 mM) is required to inhibit yeast growth, it is reported that the intracellular concentration is only about 0.1 mM (3). Since vanadate enters the cell by an active process (5), one mechanism for resistance could be a change in the transport system(s) that reduces uptake or enhances excretion. Alternatively, resistant cells could develop more effective detoxification mechanisms (2) or target site(s) could in some way become desensitized. Vanadate is known to inhibit enzymes of phosphate metabolism (2, 4), thereby affecting plasma membrane ion pumps and cytoplasmic motility (5), and it is expected that vanadate could affect processing, sorting, and secretion of macromolecules.It was recently found (R.A.H., C. Y. Chen, N. J. Simpson, and V. Chisholm, unpublished data) that the S. cerevisiae mnn9 mutant, which is defective in protein glycosylation (6), is remarkably resistant to orthovanadate and sensitive to hygromycin B, compared to the parent strain X2180. We have now analyzed a number of vanadate-resistant mutants to determine whether the glycosylation defect is a general property ofthis class. Ofthe five complementation groups we identified, all show defects in protein glycosylation and three of the groups are allelic with the previously characterized mnn8, mnn9, and mnnlO mutants (6). This result suggests that the primary sites of vanadate inhibition impinge directly on the processing of glycoproteins in the Golgi.MATERIALS AND METHODS Sodium orthovanadate, Na3VO4, was from Aldrich. Vanadate-containing plates were made by adding a freshly prepared filter-sterilized sodium orthovanadate solution to a melted sterilized agar medium, which was then poured into sterile Petri plates. Antisera that recognized the wild-type terminal al -3-linked mannose determinant and the unsubstituted al 6-linked mannose determinant of the mnn2 mutant outer chain came from laboratory stocks (7). The cell surface pheno...

The Saccharomyces cerevisiae MNTI gene encodes a Golgi mannosyltrferse. Gene disruption of the MNTI locus leads to a >90% reduction of specific a-1,2-mannosyltransferase activity with a-methyhmannoside as acceptor. Null mutants of MNTI are viable, have no apparent growth defect, and are blocked In the eloti of protein O0-nked aoblose. Structual analysis of the N-linked outer chain isolated from an mxan manlO matl train revealed no alteration In carbohydrate structure compared to the parental man) mnnlO strain. The MNTI gene is identical to KRE2, and mutations in the gene render cells resistant to the killer toxin K1 of S. cerevisiae, which suggests a role for O-mannosylated proteins in the resistance mechanism. In addition, MNTI is part of a multigene family whose members are presumed to be yast Gogi maoyltnsferases.The biosynthesis of the carbohydrate portion of glycoproteins has been studied in detail in several organisms (1, 2), and the glycosyltransferases involved in the formation of N-and O-glycans are known to be membrane proteins localized'to the endoplasmic reticulum and Golgi apparatus (3). In the yeast Saccharomyces cerevisiae, the core structure ofN-glycans is identical to that found in mammalian glycoproteins, but later modification of these core oligosaccharides in the Golgi apparatus differs considerably. In S. cerevisiae, the N-linked chain is processed by the addition of mannose from GDP-mannose to give two oligosaccharide classes, a small type with 8-14 mannoses and a large type with up to 200 mannoses, both of which may be substituted with mannosylphosphate groups (4, 5). The structure and biosynthesis of the N-linked oligosaccharides have been mainly deduced from the effects of the mnn mutations (4, 5).Less is known in yeast about the biosynthesis of the O-linked carbohydrate chains attached to serine and threonine. It has been established that the first sugar is transferred from dolichol-phosphate-mannose to serine or threonine in the endoplasmic reticulum (6, 7), and the protein-Omannosyltransferase that catalyzes this reaction has been partially purified and characterized (8, 9). Subsequently, GDP-mannose is the donor for addition of two a-1,2-linked mannoses followed by two a-1,3-linked mannoses, reactions that apparently occur in the Golgi.Despite extensive studies of the glycosylation pathways, major questions remain concerning the subcellular organization and regulation of the enzymes involved in N-and O-linked glycosylation. To address such questions, we have purified an a-1,2-mannosyltransferase from S. cerevisiae and cloned its structural gene (10). The gene, MNTI, encodes a 442-amino acid membrane-bound protein that has one N-glycosylation site and a membrane-spanning domain near its N terminus. Thus, its topology is similar to that of mammalian Golgi glycosyltransferases (3).In this report we show that Mntlp is a mannosyltransferase that is involved in adding the third mannose to the O-linked chains but that it does not appear to act on N-linked carbohydrate chains. MATERI...

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by 'H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein. The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wildtype mannoproteins.Baker's yeast carboxypeptidase Y (CPY) is a well-characterized glycoprotein (1, 2) that has assumed importance as a vacuolar marker enzyme (3) for studies on protein transport and localization (4). Its amino acid sequence, inferred, in part, from the gene sequence (5), reveals four potential glycosylation sites, which agrees with the earlier conclusion that the protein contains four N-linked oligosaccharides (6