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 Km 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.
Highlights
The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases
The excitation wavelength was set at 360 nm, the Fura-2 isosbestic point at which its properties are independent on the Ca2ϩ concentration (Fig. S1), to reflect only Mn2ϩ transport
To what was observed for the inductively coupled plasma atomic emission spectrometry (ICP–AES) analyses, expression of GDT1 under the TPI1 promoter could complement the growth defect of the gdt1⌬pmr1⌬ in the presence of manganese. These results show that, in complement to Pmr1p, Gdt1p plays a role in yeast cellular manganese homeostasis
Summary
The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. The acidic and polar uncharged amino acids of the conserved motif EXGD(K/R)(T/S) found in Gdt1p were demonstrated to be essential for proper Ca2ϩ tolerance and Ca2ϩ response to salt stress [5] In addition to this role in calcium homeostasis, recent studies on other UPF0016 members suggested an implication of these proteins in manganese homeostasis. TMEM165 and a Myc-tagged version of Gdt1p were both reported to be degraded in the presence of high extracellular manganese concentration [12, 13] These studies all established a clear functional link between the UPF0016 members and manganese homeostasis, none of them provided direct evidence for Mn2ϩ transport. This study places Gdt1p as a novel yeast Golgi protein which, together with Pmr1p, tightly regulates the Ca2ϩ and Mn2ϩ concentrations at the Golgi level
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