Investigating the function of Gdt1p in yeast Golgi glycosylation

• Published in: Biochimica et biophysica acta. General subjects Vol. 1862; no. 3; pp. 394 - 402
• Main Authors: Dulary, Eudoxie, Yu, Shin-Yi, Houdou, Marine, de Bettignies, Geoffroy, Decool, Valérie, Potelle, Sven, Duvet, Sandrine, Krzewinski-Recchi, Marie-Ange, Garat, Anne, Matthijs, Gert, Guerardel, Yann, Foulquier, François
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2018; Elsevier
• Subjects: Amino Acid Motifs; Binding Sites; Biochemistry, Molecular Biology; Ca2 + homeostasis; calcium; Calcium - metabolism; Calcium Channels - chemistry; Calcium Channels - genetics; Calcium Channels - physiology; Calcium-Transporting ATPases - metabolism; cations; Conserved Sequence; Gdt1p; Glycosylation; Golgi Apparatus - metabolism; Golgi glycosylation; homeostasis; Ion Transport; Life Sciences; manganese; Manganese - metabolism; Mannans - metabolism; Mn2 + homeostasis; Molecular Chaperones - metabolism; Monosaccharides - metabolism; mutants; nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - metabolism; Pmr1p; Protein Processing, Post-Translational - physiology; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Structural Biology; yeasts; Ca2+ homeostasis; Gdt1p; Mn2+ homeostasis; Pmr1p; Golgi glycosylation; Ca(2+) homeostasis; Mn(2+) homeostasis
• DOI: 10.1016/j.bbagen.2017.11.006
• ISSN: 0304-4165

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 gdt1Δ mutant cultured in presence of high Ca2+ concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn2+ confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca2+. The use of Pmr1p mutants either defective for Ca2+ or Mn2+ transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ 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 Pmr1p exclusively transports Ca2+. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn2+ sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. 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. •Functional link between Gdt1p and Pmr1p•Gdt1p imports Mn2+ inside the Golgi lumen when Prm1p exclusively transports Ca2+•Ion homeostasis and yeast Golgi glycosylation