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

Abstract

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn²⁺ homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis Using Mn²⁺-induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn²⁺ influx, in addition to its previously reported regulation of Ca²⁺ influx. The estimated K_m values of Gdt1p of 15.6 ± 2.6 μm for Ca²⁺ and 83.2 ± 9.8 μm for Mn²⁺ indicated that Gdt1p has a higher affinity for Ca²⁺ than for Mn²⁺ In yeast cells, we found that Gdt1p is involved in the resistance to high Mn²⁺ concentration and controls total Mn²⁺ stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn²⁺-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn²⁺ concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn²⁺ homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation.

Keywords: Congenital Disorder of Glycosylation; GDT1; Golgi; UPF0016; calcium transport; manganese; membrane transport; transporter; yeast.