The essential Smp3 protein is required for addition of the side-branching fourth mannose during assembly of yeast glycosylphosphatidylinositols

The major glycosylphosphatidylinositols (GPIs) transferred to protein in ma mmals and trypanosomes contain three mannoses, In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, alpha1,2-linked Ma n on the alpha1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-se nsitive smp3 mutants accumulate a GPI containing three mannoses and that sm p3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally res ult in the accumulation of Man(4)-GPIs, including the presumed substrate fo r the yeast GPI transamidase. The Smp3 protein, which is encoded by an esse ntial gene, is therefore required for addition of the fourth Man to yeast G PI precursors. The finding that smp3 prevents the formation of the Man(4)-G PI 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 transf er of EthN-P to Man-3 of yeast GPIs, The Man(3)-GPI that accumulates in smp 3 is a mixture of two dominant isoforms, one bearing a single EthN-P side b ranch on Man-1, the other with EthN-P on Man-2, and these isoforms can be p laced in separate arms of a branched GPI assembly pathway. Smp3-related pro teins are encoded in the genomes of Schizosaccharomyces pombe, Candida albi cans, Drosophila melanogaster, and Homo sapiens and form a subgroup of a fa mily 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 prote ins, which act in the dolichol pathway for N-glycosylation, Because Man(4)- containing GPI precursors are normally formed in yeast and Plasmodium falci parum, whereas addition of a fourth Man during assembly of mammalian GPIs i s rare and not required for GPI transfer to protein, Smp3p-dependent additi on of a fourth Man represents a target for antifungal and antimalarial drug s.