In vivo N-glycosylation of the mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus

Mol Microbiol. 2000 Nov;38(3):552-64. doi: 10.1046/j.1365-2958.2000.02151.x.

Abstract

Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression. The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family. In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation. To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the C-terminal tail of each Mep protein. When electrophoresed on SDS-polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a high-molecular-weight smear. Protein deglycosylation with peptide-N-glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine-linked glycoprotein. Site-directed mutagenesis of the four potential N-glycosylation sites of Mep2 shows that Asn-4 of the protein's N-terminal tail is the only site that binds oligosaccharides. This provides evidence for the extracytosolic location of the Mep2 N-terminus. Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N-terminal tail of Mep2. The protein's C-terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein. Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / metabolism
  • Amino Acid Sequence
  • Blotting, Western
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cation Transport Proteins*
  • Cell Membrane / metabolism
  • Cytosol / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Quaternary Ammonium Compounds / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*

Substances

  • Carrier Proteins
  • Cation Transport Proteins
  • MEP2 protein, S cerevisiae
  • Quaternary Ammonium Compounds
  • Saccharomyces cerevisiae Proteins
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase