A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

Large-scale screening of genetic and chemical-genetic interactions was used to examine the assembly and regulation of ␤-1,3-glucan in Saccharomyces cerevisiae. Using the set of deletion mutants in ‫0064ف‬ nonessential genes, we scored synthetic interactions with genes encoding subunits of the ␤-1,3-glucan synthase (FKS1, FKS2), the glucan synthesis regulator (SMI1/KNR4), and a ␤-1,3-glucanosyltransferase (GAS1). In the resulting network, FKS1, FKS2, GAS1, and SMI1 are connected to 135 genes in 195 interactions, with 26 of these genes also interacting with CHS3 encoding chitin synthase III. A network core of 51 genes is multiply connected with 112 interactions. Thirty-two of these core genes are known to be involved in cell wall assembly and polarized growth, and 8 genes of unknown function are candidates for involvement in these processes. In parallel, we screened the yeast deletion mutant collection for altered sensitivity to the glucan synthase inhibitor, caspofungin. Deletions in 52 genes led to caspofungin hypersensitivity and those in 39 genes to resistance. Integration of the glucan interaction network with the caspofungin data indicates an overlapping set of genes involved in FKS2 regulation, compensatory chitin synthesis, protein mannosylation, and the PKC1-dependent cell integrity pathway. al. 2002). In addition to the Rho1p regulatory subunit, other proteins are required for normal levels of ␤-1,3-cells, is responsible for cell shape and osmotic stability, and acts as a filter for large molecules. The cell wall glucan. The SMI1/KNR4 gene was cloned by complementation of a Hansenula mrakii K9 killer toxin (a glucan is composed mainly of ␤-1,3 and ␤-1,6-glucans, mannoproteins, and chitin, with the relative proportions of synthase inhibitor) resistant mutant (Hong et al. 1994). T HE cell wall is a major organelle that surroundsThe smi1⌬ mutant has a highly permeable cell wall and these constituents varying with growth conditions and shows both decreased glucan synthase activity and cell the cellular developmental program. ␤-1,3-Glucan is the wall ␤-1,3-glucan content (Hong et al. 1994; Martin et principal cell wall component, to which the other comal. 1999). Genetic and biochemical evidence suggests ponents are crosslinked (Smits et al. 1999; that Smi1p acts in the PKC1-SLT2 signaling cascade by 2002). Synthesis of ␤-1,3-glucan occurs at the plasma modulating the kinase activity of Slt2p (Martin-Yken membrane. Glucan synthase is thought to contain a et al. 2002, 2003). catalytic subunit, encoded by the two homologous genes Cell wall composition changes during growth, bud-FKS1 and FKS2/GSC2 (Mazur et al. 1995), and a regulading, mating, and sporulation, and these dynamic protory subunit, the small GTPase Rho1p (Drgonova et cesses require remodeling of the crosslinking of ␤-1, 3-al. 1996; Mazur and Baginsky 1996; Qadota et al.and ␤-1,6-glucans to themselves and to other cell wall 1996). FKS1 and FKS2 encode a pair of integral membrane components. Gas1p, a GPI-anchored protein localized proteins wi...

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Abstract. The yeast Kre2p/Mntlp cxl,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mntlp antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNNl-dependent manner. Kre2p/Mntlp is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnnlp, and distinct from that including the late Golgi protein Kexlp. To determine which regions of Kre2p/Mntlp are required for Golgi localization, Kre2p/Mntlp mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The resuits demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36--amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi. THE Golgi apparatus plays a fundamental role in glycan processing and sorting of newly synthesized proteins in the secretory pathway of eukaryotic cells. The Golgi apparatus of a typical mammalian cell is composed of a polarized stack of membranous saccules that are differentiated into functionally distinct subcompartments. After the addition of core N-linked sugar moieties in the endoplasmic reticulum, glycoproteins proceed through the cis-Golgi network, the cis-, medial-, and transcisternae, and the trans-Golgi network, where further modifications take place. These include the addition of O-linked sugars and the elaboration, in a protein-specific manner, of complex N-linked carbohydrate structures. The Golgi apparatus also constitutes a major organelle responsible for protein trafficking where particular proteins are directed to precise cellular locations. Two specific Golgi subcompartments have been found to be involved in protein sorting. The cis-Golgi network constitutes the site from which many resident ER proteins are retrieved, and the main function of the TGN is to direct glycoproteins exiting the Golgi complex to either the lysosome or the cell surface

Nascent proteins synthesized by membrane-bound ribosomes are translocated across the ER 1 membrane and acquire carbohydrate chains on specific serine, threonine, and asparagine residues. These glycoproteins then proceed through the Golgi complex where their oligosaccharides are further modified. The O-modified proteins of Saccharomyces cerevisiae possess a linear carbohydrate chain of up to 5 mannose residues (1,2). O-Glycosylation is initiated in the ER with dolichyl phosphate-D-mannose as the immediate sugar donor for the mannosyl residue transferred to the hydroxy-amino acids serine and threonine (2, 3). GDP-Man is utilized as the sugar donor in the subsequent elongation of O-linked carbohydrate chains resulting in a linear glycan in which the second and third mannoses possess ␣1,2-linkages, whereas the terminal fourth and fifth mannosyl residues have ␣1,3-linkages (1, 2).Yeast N-linked modified proteins can acquire two different types of glycan chains after the addition of a Man 8 GlcNAc 2 core in the endoplasmic reticulum, a process common to the majority of eukaryotes (1, 2). This primary oligosaccharide can undergo maturation in the Golgi resulting in a Man 8 -13 GlcNAc 2 carbohydrate structure, or it may be extended by an outer chain of variable size (up to 200 mannose residues) that is made up of a backbone of ␣1,6-mannosyl residues to which are attached branched ␣1,2-and ␣1,3-mannosyl side chains.Some of the enzymes involved in the elaboration of O-linked oligosaccharides and in the synthesis of N-linked outer chains have been identified, and their structural genes have been isolated. At least four different genes of the seven-membered PMT1-7 gene family encoding dolichyl phosphate-D-mannose: protein O-D-mannosyltransferases are responsible for initiating the O-linked glycans (4). Protein O-glycosylation is essential for cell function because mutants of S. cerevisiae lacking different combinations of three of the PMT genes are not viable (4). The OCH1 gene is responsible for adding the first ␣1,6-mannose residue involved in initiating N-linked outer chain elaboration (5, 6). Two enzymes in particular have been shown to participate in both O-and N-linked glycosylation. The KRE2/ MNT1 gene encodes a medial Golgi ␣1,2-mannosyltransferase required for the addition of the third mannose residue on O-linked chains (7,8) and is also implicated in N-linked outer chain oligosaccharide synthesis (9, 10). The O-linked trisaccharide can be further extended by one or two ␣1,3-linked mannoses. The fourth mannose residue is added by the Golgi localized ␣1,3-mannosyltransferase Mnn1p, which also terminally mannosylates core and outer chain modified N-linked glycans (8,(11)(12)(13).To further characterize protein glycosylation in yeast and identify some of the responsible glycosyltransferases, we have studied a gene family encoding proteins that are highly homologous to Kre2p/Mnt1p. This nine-membered KTR mannosyltransferase gene family contains the KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6,. As with other kno...

Background: In S. cerevisiae the β-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants.

Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1-MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-beta-glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZEO1 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP-GTP exchange factor Rom2p, while exacerbating the growth defect of sac7delta mutants at 37 degrees C. In contrast, mid2delta mutants have opposing effects to zeo1delta mutants, being synthetically lethal with rom2delta, and suppressing an 18 degrees C growth defect of sac7delta, while overexpression of MID2 rescues a rom2delta 37 degrees C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1-MPK1 cell integrity pathway.

Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying a yeast gene family in which at least one member, KRE2/MNT1, is involved in protein glycosylation. The family currently contains five other members: YUR1, KTR1, KTR2, KTR3 and KTR4 (Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). All encode putative type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. Kre2p/Mnt1p is a alpha 1,2-mannosyltransferase involved in O- and N-linked glycosylation (Häusler, A., Ballou, L., Ballou, C.E., and Robbins, P.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6846-6850); however, the role of the other proteins has not yet been established. We have carried out a functional analysis of Ktr1p, Ktr2p, and Yur1p. By in vitro assays, Ktr1p, Ktr2p, and Yur1p have been shown to be mannosyltransferase but, in vivo, do not appear to be involved in O-glycosylation. Examination of the electrophoretic mobility of the N-linked modified protein invertase in null mutant strains indicates that Ktr1p, Ktr2p, and Yur1p are involved in N-linked glycosylation, possibly as redundant enzymes. As found with Kre2p (Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H. (1992) Genetics 130, 273-283), Ktr1p, Ktr2p, and Yur1p also seem to be implicated in the glycosylation of cell wall mannoproteins, since yeast cells containing different gene disruptions become K1 killer toxin-resistant. Immunofluorescence microscopy reveals that like Kre2p; Ktr1p, Ktr2p and Yur1p are localized in the Golgi complex.