The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, ␣1,2-linked Man on the ␣1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperaturesensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man 4 -GPIs, including the presumed substrate for the yeast GPI transamidase. The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors. The finding that smp3 prevents the formation of the Man 4 -GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs. The Man 3 -GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway. Smp3-related proteins are encoded in the genomes of Schizosaccharomyces pombe, Candida albicans, Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation. Because Man 4 -containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs.

Glycosylphosphatidylinositols (GPIs) are found in all eukaryotes and are synthesized in a pathway that starts with the transfer of N-acetylglucosamine (GlcNAc) from UDPGlcNAc to phosphatidylinositol (PI). This reaction is carried out by a protein complex, three of whose subunits in humans, hGpi1p, Pig-Cp and Pig-Ap, have sequence and functional homologues in the Saccharomyces cerevisiae Gpi1, Gpi2 and Gpi3 proteins, respectively. Human GlcNAc-PI synthase contains two further subunits, Pig-Hp and PigPp. We report that the essential YNL038w gene encodes the S. cerevisiae homologue of Pig-Hp. Haploid YNL038w-deletion strains were created, in which Ynl038wp could be depleted by repressing YNL038w expression using the GAL10 promoter. Depletion of Ynl038wp from membranes virtually abolished in vitro GlcNAc-PI synthetic activity, indicating that Ynl038wp is necessary for GlcNAc-PI synthesis in vitro. Further, depletion of Ynl038wp in an smp3 mutant background prevented the formation of the trimannosylated GPI intermediates that normally accumulate in this late-stage GPI assembly mutant. Ynl038wp is therefore required for GPI synthesis in vivo. Because YNL038w encodes a protein involved in GPI biosynthesis, we designate the gene GPI15. Potential Pig-Hp/Gpi15p counterparts are also encoded in the genomes of Schizosacchomyces pombe and Candida albicans.

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI). The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex. Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins. To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains. In Sacch. cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner. In addition, in vitro GlcNAc-PI synthetic activity was again detectable. In Schiz. pombe, gpi1+ disruption is lethal. Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele. Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex. The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution.

The objectives of this study were to determine if there were variations in triazine-resistant and -susceptible Brassica napus leaf disc chlorophyll fluorescence (LCF) intensity in terms of: age of leaf on the plant and time of day. In growth room and field experiments triazine-susceptible B. napus cv. “Tower”, and triazine-resistant B. napus cv. “OAC Triton” were used. Chlorophyll fluorescence intensity measurements were made 30 min after disc removal. In both environments, two periods of reduced photosynthetic efficiency occurred in the diurnal. The times that these periods occurred during the diurnal differed between biotypes. A phase shift in LCF maxima between resistant and susceptible biotypes resulted in two periods, early and late in the light period of the diurnal, of increased LCF in resistant tissue. This differential pattern in LCF is support for the hypothesis that triazine resistance chloroplast alterations could imply an alteration in the temporal organization of chloroplast physiological function.

No abstract