Investigating the function of Gdt1p in yeast Golgi glycosylation

Abstract

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 Ca/Mn homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca 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 Mn 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 Ca. The use of Pmr1p mutants either defective for Ca or Mn transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn inside the Golgi lumen when Pmr1p exclusively transports Ca. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn 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 Mn/Catransport.

The yeast protein Gdt1p transports Mn2+ ions and thereby regulates manganese homeostasis in the Golgi

“…More generally, according to this hypothesis of direction of transport, Gdt1p would provide the Golgi with calcium and manganese cations and/or detoxify the cytosol in case of excess of these cations, in complement to Pmr1p. Based on the evaluation of the glycosylation process efficiency as an indicator of the Golgi Mn 2ϩ concentration, the same direction of manganese transport was suggested by Dulary et al (13). The entry of Mn 2ϩ in the Golgi would then be controlled by an ATPase, Pmr1p, and by a secondary transporter, Gdt1p.…”

“…Indeed, Colinet et al (4) previously reported Gdt1p-dependent glycosylation defects in yeast. More recently, Dulary et al (13) analyzed the structural glycosylation abnormalities observed in a gdt1⌬ strain cultured in the presence of a high external calcium concentration and reported defects in the ␣-1,3and ␣-1,2-mannose branchings that correspond to deficiencies in late Golgi glycosyltransferases like Mnn2, Mnn5, and Mnn1. Interestingly, these three mannosyltransferases all require Mn 2ϩ as a cofactor (18,20).…”

“…Manganese cations were also demonstrated to restore glycosylation in TMEM165-deficient cells (11). Interestingly, TMEM165 and a Myc-tagged version of Gdt1p were both reported to be degraded in the presence of high extracellular manganese concentration (12,13). However, although these studies all established a clear functional link between the UPF0016 members and manganese homeostasis, none of them provided direct evidence for Mn 2ϩ transport.…”

“…By now, the P-type ATPase Pmr1p is the only reported yeast Golgi transporter of Ca 2ϩ and Mn 2ϩ . By analyzing the contribution of Pmr1p and Gdt1p in the Golgi glycosylation process, Dulary et al (13) suggested that the abundance and function of Gdt1p depends on the function of Pmr1p, thereby creating a functional link between these two Golgi proteins. Our study highly reinforces the hypothesis that Gdt1p and Pmr1p act in concert in the regulation of the yeast Ca 2ϩ and Mn 2ϩ homeostasis.…”

Anomalies congénitales de la glycosylation (CDG)

“…Gdt1p[42][43][44], ces travaux ont permis d'établir un lien entre homéostasie golgienne du Mn 2+ et processus de glycosylation. TMEM165 est en effet un acteur majeur de la régulation de l'homéostasie du Mn 2+ en permettant son import dans l'appareil de Golgi(Figure 5).…”