The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these ‘single-domain membrane proteins’ some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.
Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca2+ exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells.
Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 (uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca2+- and Mn2+-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis. Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca2+ and Mn2+ transporters abrogates Ca2+- and Mn2+-induced growth defects, excessive Mn2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca2+ and Mn2+ influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca2+ and Mn2+, which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations.
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.
The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E‐φ‐G‐D‐[KR]‐[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site‐directed mutagenesis, we generated 17 mutations in the yeast Golgi‐localized Ca2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+‐ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.
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