Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca2+/Mn2+ homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdtl Delta mutant cultured in presence of high Ca2+ concentration, as well as the pmr1 Delta and gdt1 Delta/pmr1 Delta strains presented strong late Golgi glycosylation defects with a lack of alpha-1,2 mannoses substitution and alpha-1,3 mannoses termination. The addition of Mn2+ confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmrld could also completely be suppressed by the addition of Ca2. The use of Pmr1 Delta mutants either defective for Ca2+ or Mn2+ transport or both revealed that the suppression of the observed glycosylation defect in pmr1 Delta strains by the intraluminal Golgi Ca2+ requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn2+ inside the Golgi lumen when Pmrlp exclusively transports Ca2+. Our results also reinforce the functional link between Gdt1p and Pmrlp as we highlighted that Gdt1p was a Mn2+ sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmrlp. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D- [KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn2+/Ca2+ transport.