N-glycosylation is an essential co- and post- translational modification that affects protein folding, stability and function. N-glycosylation is catalyzed by an enzyme complex known as the oligosaccharyltransferase (OTase). Ost3 and Ost6 proteins are subunits of the OTase and found to have different protein substrate binding affinities1. In humans, the orthologues of OST3 and OST6 are MagT1 and TUSC3. MagT1 and TUSC3 have been found to transport magnesium as well as assist in glycosylation, hence we hypothesized that Ost3 and or Ost6 proteins could perform similar functions.
Yeast is a model organism where gene manipulation can be used to study protein function. Here, we used a Tet repressible promoter system to achieve a double knockdown/out of OST3/6 in a yeast strain. Recent advances in mass spectrometry allowed us to study proteins and their modifications extensively. In this study we employed both glyco- and global proteomics to elucidate Ost3/6 function.
We found that Ost3- and Ost6-deficient yeast had a severe growth defect, which was partially rescued by the addition of magnesium. This supports a role for Ost3 proteins in Mg2+ transport in yeast, as well as in vertebrates. However, glyco-proteomics did not show any sign of improvement in site specific glycosylation. This result suggested that Ost3 protein-mediated Mg2+ transport is independent of glycosylation. Consistent with this, global proteomics revealed extensive changes in ribosomal proteins, metabolic processes and translation. Of these changes, ribosomal proteins were rescued by the addition of excess Mg2+.
The dual function of Ost3 proteins in regulating N-glycosylation and in transporting Mg2+ appears to be conserved from vertebrates to fungi. Ost3 protein-mediated Mg2+ transport is required for normal cellular levels of ribosomes, potentially due to the direct stabilizing effect of Mg2+ on intact ribosomes.