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 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 Mn homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Using Mn-induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn influx, in addition to its previously reported regulation of Ca influx. The estimated values of Gdt1p of 15.6 ± 2.6 μm for Ca and 83.2 ± 9.8 μm for Mn indicated that Gdt1p has a higher affinity for Ca than for Mn In yeast cells, we found that Gdt1p is involved in the resistance to high Mn concentration and controls total Mn stores. Lastly, we demonstrated that deletion affects the activity of the yeast Mn-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn 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 Mn 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.

Milk biosynthesis requires the Golgi cation exchanger TMEM165

“…The residual partial defect in glycosylation in gdt1⌬ yeast cells was exacerbated by elevated Ca 2ϩ and then ameliorated by supplementation with Mn 2ϩ in a fashion that depended on the SPCA known as Pmr1 (3). Furthermore, when heterologously expressed in bacteria, Gdt1 has been demonstrated to transport Mn 2ϩ , although it exhibited a lower affinity for Mn 2ϩ than for Ca 2ϩ (4). Presumably, the mechanism of Mn 2ϩ transport by Gdt1 is similar to that of Ca 2ϩ , which has been demonstrated to be coupled to the pH gradient established by the V-ATPase, the deletion of which causes Gdt1 to function in reverse mode, instead releasing Ca 2ϩ from Golgi lumen (29).…”

“…19). Because TMEM165 deficiency is known to cause congenital disorders of glycosylation (4,20), it was hypothesized that disruption of…”

“…Deficiencies in the Golgi transmembrane protein TMEM165 cause a type II congenital disorder of glycosylation in humans (1). Recent reports have shown that this protein, along with its yeast homolog Gdt1, can transport Ca 2ϩ in vitro and promote normal homeostasis of Ca 2ϩ , Mn 2ϩ , and H ϩ in the Golgi complex (2)(3)(4). These findings suggest that TMEM165 supports glycosylation by maintaining proper pH and supplying Mn 2ϩ and Ca 2ϩ that are required as cofactors in the glycosyltrans-ferases responsible for protein and lipid glycosylation (5,6).…”

Bacillus subtilisTerC Family Proteins Help Prevent Manganese Intoxication

“…More generally, TerC proteins are related to the larger UPF0016 family of proteins, which includes the plant PAM71 protein, postulated to translocate Mn(II) into the chloroplast lumen for photosystem assembly (58). Similarly, the yeast UPF0016 protein Gdt1p translocates Mn(II) and Ca(II) from the cytosol to the Golgi compartment, where it contributes to activities required for protein glycosylation (59). It is interesting to note that when expressed in the bacterium Lactococcus lactis, yeast Gdt1p mediated Mn(II) influx.…”

“…Although we favor a model in which bacterial TerC homologs function physiologically in Mn(II) export, perhaps coupled to metallation of secreted or membrane-localized enzymes, they will likely function in import if inserted in the membrane in the wrong orientation. This could explain the impact of expressing a heterologous yeast protein in L. lactis (59) and may also account for the reported increase in cytosolic Mn(II) when the E. coli Alx protein was overproduced (42). Further studies will be required to test this general hypothesis regarding the possible roles of TerC proteins in B. subtilis physiology.…”