Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca 2+ exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca 2+ homeostasis via an uncharacterized Ca 2+ transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca 2+ , and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca 2+ ion transport. Furthermore, defects in TMEM165 affected both Ca 2+ and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca 2+ /H + antiporters and that modification of the Golgi Ca 2+ and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients. CDGs are a family of inborn metabolic diseases affecting the glycosylation pathway. Most of these mutations are found in genes directly involved in glycosylation, however unique types of CDG have been found to be caused by deficiencies in vesicular Golgi trafficking (2-6) and Golgi pH homeostasis (7). TMEM165 belongs to a well-conserved, but uncharacterized, family of membrane proteins named UPF0016 (Uncharacterized Protein Family 0016; Pfam PF01169) and is localized in the Golgi apparatus (1). The members of this family are well conserved and are found in many organisms-for example, 919 different species of bacteria and 409 different eukaryotes.Gcr1 dependent translation factor 1 (Gdt1p) the yeast ortholog of TMEM165, is a 280-residue membrane protein and is involved in tolerance to high concentrations of calcium (Ca 2+ ) (8). In eukaryotic cells, Ca 2+ is a ubiquitous intracellular messenger involved in many different biological processes (9). To allow the increases in cytosolic calcium concentration ([Ca 2+ ] cyt ) required for these signaling mechanisms, it is absolutely necessary that the resting Ca 2+ levels are maintained below a certain threshold. Under normal conditions, the yeast [Ca 2+ ] cyt is maintained between 50 and 200 nM (10). The maintenance of this basal level and the return to normal levels after stimulation are achieved by a series of Ca 2+ pumps and exchangers located in different compartments of the cell: Pmr1p, the P-type Ca 2+ / Mn 2+ -ATPase localized in the medial-Golgi apparatus and responsible for the Ca 2+ supply for the secretory pathway (11-13), and Pmc1p (14), a P-type Ca 2+ -ATPase, and Vcx1p (15, 16), a Ca 2+ /H + exchanger (CAX), both responsible for the uptake of Ca 2+ through the vacuolar membrane.Yeast is a simple and conv...
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.
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.
The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis-and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.
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