Structure-Function Analysis of the Dolichyl Phosphate-Mannose: Protein O-Mannosyltransferase ScPmt1p

Abstract

Protein O-mannosylation is an essential protein modification. It is initiated at the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to humans. Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the endoplasmic reticulum. ScPmt1p forms a complex with ScPmt2p that is required for maximum transferase activity. Recently, we proposed a seven-transmembrane structural model for ScPmt1p. A large, hydrophilic, endoplasmic reticulum-oriented segment is flanked by five aminoterminal and two carboxyl-terminal membrane-spanning domains. Based on this model, a structure-function analysis of ScPmt1p was performed. Deletion mutagenesis identified the N-terminal third of the transferase as being essential for the formation of a functional ScPmt1p-ScPmt2p complex. Deletion of the central hydrophilic loop eliminates mannosyltransferase activity, but not ScPmt1p-ScPmt2p interactions. Alignment of all fully characterized PMT family members revealed that this central loop region contains three highly conserved peptide motifs, which can be considered as signatures of the PMT family. In addition, a number of invariant amino acid residues were identified throughout the entire protein sequence. In order to evaluate the functional significance of these conserved residues site-directed mutagenesis was performed. We show that several amino acid substitutions in the conserved motifs significantly reduce ScPmt1p activity. Further, the invariant residues Arg-64, Glu-78, Arg-138, and Leu-408 are essential for ScPmt1p function. In particular, Arg-138 is crucial for ScPmt1p-ScPmt2p complex formation.Protein O-mannosylation is of fundamental importance in numerous physiological processes. In the yeast Saccharomyces cerevisiae, O-mannosylation is an essential protein modification that is indispensable for cell morphology and cell wall integrity (1, 2). Furthermore, O-linked polymannose chains are required for the stability and/or correct localization of proteins (3, 4) and also affect protein function (5, 6). In the opportunistic pathogenic fungus Candida albicans, reduced protein O-mannosylation results in defects in morphogenesis, abated adherence to host cells, and a strong attenuation of virulence in mouse model systems (7, 9). 1In yeasts and filamentous fungi, O-linked mannooligosaccharide synthesis is initiated in the endoplasmic reticulum (ER) 2 by the transfer of a mannosyl residue from dolichyl phosphate-activated mannose (Dol-P-Man) to specific serine/ threonine residues of proteins entering the secretory pathway (2). Further modification of the O-linked mannose takes place in the Golgi apparatus (2, 10).Dol-P-Man:protein O-mannosyltransferases (Pmts) comprise a large family of enzymes that initiate protein O-mannosylation at the ER. In Saccharomyces cerevisiae, the seven members of this protein family (ScPmt1p to ScPmt7p) feature an overall sequence similarity of 50 -55% and, more strikingly, a nearly identical hydropathy p...

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

“…This was confirmed by cross-linking m⌬gp␣f to Sec61p ( Figure 4B). The Pmts in the ER are polytopic transmembrane proteins with large ER-luminal domains containing the active sites (Girrbach et al, 2000). It is therefore conceivable that Pmts modify membrane-associated substrates more efficiently; Pmts may even be located in close proximity to the translocon, which would ensure that wild-type secretory proteins with O-mannosyl acceptor sites are modified efficiently and without interference from protein folding during entry into the ER through the Sec61 channel.…”

“…The individual transferases show distinct specificities toward their protein substrates Tanner, 1996, 1997). The active sites of the Pmts are predicted to be on the luminal face of the ER membrane (Girrbach et al, 2000). The transferases Pmt1p and Pmt2p have been shown to act as in the presence of ATP and an ATP-regenerating system, cytosol or both.…”

“…heterodimers in vitro and in vivo (Girrbach et al, 2000). We prepared microsomes from all viable pmt1-4 double and triple mutants and from strains with individual deletions of PMT5 and PMT6 (Gentzsch and Tanner, 1996); we subsequently translocated p⌬gp␣f into wild-type and pmt mutant microsomes for 50 min at 24°C, lysed the membranes, and assessed ⌬gp␣f mannosylation by precipitation with ConASepharose.…”

The Twisted Abdomen Phenotype of Drosophila POMT1 and POMT2 Mutants Coincides with Their Heterophilic Protein O-Mannosyltransferase Activity

“…In the yeast Saccharomyces cerevisiae, O-linked oligomannose chains are required for the stability, correct localization, and/or function of proteins (1)(2)(3)(4)(5)(6). Yeast O-mannosylation is initiated in the lumen of the endoplasmic reticulum by a family of protein O-mannosyltransferases, PMT1-PMT6, 1 which catalyze the transfer of Man from dolichylphosphate Man to Ser or Thr residues of secretory proteins (7)(8)(9). The PMT family is classified phylogenetically into the PMT1, PMT2, and PMT4 subfamilies.…”

Characterization of Ccw12p, a major key player in cell wall stability of Saccharomyces cerevisiae

“…Yeast cells were grown in SD medium to 2 × 10 7 cells/ml, harvested, and crude membranes were prepared as described in Girrbach et al (2000).…”