The Essential Smp3 Protein Is Required for Addition of the Side-branching Fourth Mannose during Assembly of Yeast Glycosylphosphatidylinositols

Abstract

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.

Human Smp3p Adds a Fourth Mannose to Yeast and Human Glycosylphosphatidylinositol Precursors in Vivo

“…Locus 80235 of human chromosome 3 encodes a predicted 579-amino acid protein (FLJ12769) that has been grouped into a family of dolichol phosphate mannose-utilizing mannosyltransferase sequences by its homology with the asparagine glycosylation pathway mannosyltransferases Alg9p and Alg12p and the GPI mannosyltransferases PIG-Bp and S. cerevisiae Smp3p (23,36). Human FLJ12769 contains a HQEXRF amino acid motif that is characteristic of the SMP3 group within this sequence family.…”

“…In S. cerevisiae, late stage intermediates in GPI precursor synthesis (17, 19 -21), the presumed GPI transamidase substrates (5), and protein-bound GPIs (22) all contain four mannoses. Addition of Man-4 to trimannosyl-GPIs (Man 3 -GPIs) by the essential Smp3 mannosyltransferase is a mandatory step in yeast GPI precursor assembly which precedes the addition of the terminal EthN-P to Man-3 through which the GPI is ultimately attached to protein (23). Thus, it is probable that all yeast GPI transamidase substrates bear four mannoses.…”

Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II ofSaccharomyces cerevisiae

“…To test the possibility that Pga1 has genetic interaction with other GPI-MTs, we introduced GPI14, PBN1, GPI10, or SMP3 on 2 plasmid. GPI14 and PBN1 encode subunits of GPI-MT-I (Maeda et al, 2001;Ashida et al, 2005), GPI10 encodes the third ␣1,2-mannosyltransferase, and SMP3 encodes the last ␣1,2-mannosyltransferase (Grimme et al, 2001). However, multicopy expression of each gene could not restore the growth of the pga1-1 mutant at 37°C ( Figure 6B).…”

“…The enzymes that add Man to GPI precursors are called GPI-mannosyltransferase (GPI-MT) I, -II, -III, and -IV, according to the order of mannose addition. Each GPI-MT consists of at least one protein with several transmembrane domains that has homology to other enzymes responsible for glycosylation (Takahashi et al, 1996;Grimme et al, 2001;Maeda et al, 2001;Ashida et al, 2005;Fabre et al, 2005;Kang et al, 2005). So far, GPI-MT I has been the only enzyme that is found to consist of multisubunits.…”

“…The fourth Man has essential roles because SMP3, which encodes the enzyme to add the fourth Man, is essential for viability (Grimme et al, 2001), although the addition of fourth Man is not essential and the GPIs contain only three Man residues in other organisms (Taron et al, 2004). The enzymes that add Man to GPI precursors are called GPI-mannosyltransferase (GPI-MT) I, -II, -III, and -IV, according to the order of mannose addition.…”