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Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn‐related diseases in the future. [ABSTRACT FROM AUTHOR]

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG. [ABSTRACT FROM AUTHOR]

TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165’s function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn2+, with an extra reported function in Ca2+ and/or H+ transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca2+ signaling in yeast, and Mn2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles.

Background: The presence of portal vein tumor thrombus (PVTT) is a significant indicator of advanced-stage hepatocellular carcinoma (HCC). Unfortunately, the prediction of PVTT occurrence remains challenging, and there is a lack of comprehensive research exploring the underlying mechanisms of PVTT formation and its association with immune infiltration., Methods: Our approach involved analyzing single-cell sequencing data, applying high dimensional weighted gene co-expression network analysis (hdWGCNA), and identifying key genes associated with PVTT development. Furthermore, we constructed competing endogenous RNA (ceRNA) networks and employed weighted gene co-expression network analysis (WGCNA), as well as three machine-learning techniques, to identify the upstream regulatory microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) of the crucial mRNAs. We employed fuzzy clustering of time series gene expression data (Mfuzz), gene set variation analysis (GSVA), and cell communication analysis to uncover significant signaling pathways involved in the activation of these important mRNAs during PVTT development. In addition, we conducted immune infiltration analysis, survival typing, and drug sensitivity analysis using The Cancer Genome Atlas (TCGA) cohort to gain insights into the two patient groups under study., Results: Through the implementation of hdWGCNA, we identified 110 genes that was closely associated with PVTT. Among these genes, TMEM165 emerged as a crucial candidate, and we further investigated its significance using COX regression analysis. Furthermore, through machine learning techniques and survival analysis, we successfully identified the upstream regulatory miRNA ( hsa-miR-148a ) and lncRNA ( LINC00909 ) that targeted TMEM165 . These findings shed light on the complex regulatory network surrounding TMEM165 in the context of PVTT. Moreover, we conducted CIBERSORT analysis, which unveiled correlations between TMEM165 and immune infiltration in HCC patients. Specifically, TMEM165 exhibited associations with various immune cell populations, including memory B cells and CD8 + T cells. Additionally, we observed implications for immune function, particularly in relation to immune checkpoints, within the context of HCC., Conclusions: The regulatory axis involving TMEM165 , hsa-miR-148a , and LINC00909 emerges as a crucial determinant in the development of PVTT in HCC patients, and it holds significant implications for prognosis. Furthermore, alterations in the TMEM165 / hsa-miR-148a / LINC00909 regulatory axis exhibit a strong correlation with immune infiltration within the HCC tumor microenvironment, leading to immune dysfunction and potential failure of immunotherapy., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-23-1589/coif). The authors have no conflicts of interest to declare., (2024 Translational Cancer Research. All rights reserved.)

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn 2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro‐α‐trypsin inhibitor and inter‐α‐trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western‐blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V‐ATPase (ATP6V0A2‐CDG, CCDC115‐CDG, ATP6AP1‐CDG), in Golgi manganese homeostasis (TMEM165‐CDG) and in the N‐acetyl‐glucosamine Golgi transport (SLC35A3‐CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in‐depth characterized by two‐dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases. [ABSTRACT FROM AUTHOR]

TMEM165 is a Golgi protein whose deficiency causes a Congenital Disorder of Glycosylation (CDG). We have demonstrated that Mn2+ supplementation could suppress the glycosylation defects observed in TMEM165-deficient cells and that TMEM165 was a Mn2+-sensitive protein. In the Golgi, the other transmembrane protein capable to regulate Mn2+/Ca2+ homeostasis is SPCA1, encoded by the ATP2C1 gene. A loss of one copy of the ATP2C1 gene leads to Hailey-Hailey Disease (HHD), an acantholytic skin disorder in Humans. Our latest results suggest an unexpected functional link between SPCA1 and TMEM165. In order to clarify this link in case of partial SPCA1 deficiency, HHD fibroblasts were used to assess TMEM165 expression, subcellular localization and Mn2+-induced degradation. No differences were observed regarding TMEM165 expression and localization in HHD patients' fibroblasts compared to control fibroblasts. Nevertheless, we demonstrated both for fibroblasts and keratinocytes that TMEM165 expression is more sensitive to MnCl 2 exposure in HHD cells than in control cells. We linked, using ICP-MS and GPP130 as a Golgi Mn2+ sensor, this higher Mn2+-induced sensitivity to a cytosolic Mn accumulation in MnCl 2 supplemented HHD fibroblasts. Altogether, these results link the function of SPCA1 to the stability of TMEM165 in a pathological context of Hailey-Hailey disease. • Functional link between SPCA1 and TMEM165 in Hailey-Hailey Disease. • Higher Mn2+-induced TMEM165 degradation in Hailey-Hailey Disease. • Stability of TMEM165 in Hailey-Hailey Disease. [ABSTRACT FROM AUTHOR]

TMEM165 is involved in a rare genetic human disease named TMEM165‐CDG (congenital disorders of glycosylation). It is Golgi localized, highly conserved through evolution and belongs to the uncharacterized protein family 0016 (UPF0016). The use of isogenic TMEM165 KO HEK cells was crucial in deciphering the function of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor of many glycosylation enzymes. Severe Golgi glycosylation defects are observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells and are rescued by exogenous manganese supplementation. Intriguingly, we demonstrate in this study that the observed Golgi glycosylation defect mainly depends on fetal bovine serum, particularly its manganese level. Our results also demonstrate that iron and/or galactose can modulate the observed glycosylation defects in TMEM165 KO HEK cells. While isogenic cultured cells are widely used to study the impact of gene defects on proteins' glycosylation patterns, these results emphasize the importance of the use of validated fetal bovine serum in glycomics studies. [ABSTRACT FROM AUTHOR]

Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/Mn2+ transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn2+ sensitivity. Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn2+ sensitivity. • TMEM165 is involved in Golgi glycosylation. • TMEM165 is highly sensitive to Mn2+concentration. • The UPF0016 amino acids are crucial for TMEM165 in Golgi glycosylation and Mn2+ sensitivity. [ABSTRACT FROM AUTHOR]

Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification. • Glycosylation defects in TMEM165-depleted cells arise from a lack of secretory pathway/organelle Mn homeostasis. • TMEM165 is a key protein in cellular Mn homeostasis. • Mn-detoxifying capacities of cells through the activity of SPCA1 depends on TMEM165 degradation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca 2 + /H + antiporters regulating Ca 2 + and pH homeostasis and mainly localized in the Golgi apparatus. Methods RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. Results In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. General significance The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations. [ABSTRACT FROM AUTHOR]

Congenital Disorders of Glycosylation (CDG) are rare inherited diseases causing glycosylation defects responsible for severe growth and psychomotor retardations in patients. Whereas most genetic defects affect enzymes directly involved in the glycosylation process, like glycosyltransferases or sugar transporters, recent findings revealed the impact of gene mutations on proteins implicated in both Golgi vesicular trafficking and ion homeostasis. TMEM165 is one of these deficient Golgi proteins found in CDG patients whose function in the secretory pathway has been deduced from several recent studies using TMEM165 deficient mammalian cells or yeast cells deficient in Gtd1p, the yeast TMEM165 ortholog. These studies actually confirm previous observations based on both sequence and predicted topology of this transmembrane protein and the phenotypes of human and yeast cells, namely that TMEM165 is very probably a transporter involved in ion homeostasis. Whereas the exact function of TMEM165 remains to be fully characterized, several studies hypothesize that TMEM165 could be a Golgi localized Ca 2+ /H + antiporter. However, recent data also support the role of TMEM165 in Golgi Mn 2+ homeostasis then arguing for a putative role of Mn 2+ transporter for TMEM165 essential to achieve the correct N-glycosylation process of proteins in the secretory pathway. This manuscript is a review of the current state of knowledge on TMEM165 deficiencies in Congenital Disorders of Glycosylation as well as new data on function of TMEM165 and some speculative models on TMEM165/Golgi functions are discussed. [ABSTRACT FROM AUTHOR]

TMEM165 is a Golgi protein playing a crucial role in Mn 2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn 2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R 304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R 304 mutation on TMEM165's function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: FOULQUIER reports financial support was provided by French National Research Agency., (© 2023 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Highlights: [•] TMEM165 expression pattern correlates with mammary calcium transport needs. [•] TMEM165 was found to be primarily Golgi located in the lactating mammary gland. [•] TMEM165 compensatory upregulation in PMCA2 knockouts supports mammary calcium transport function. [ABSTRACT FROM AUTHOR]

About half of the eukaryotic proteins bind biometals that participate in their structure and functions in virtually all physiological processes, including glycosylation. After reviewing the biological roles and transport mechanisms of calcium, magnesium, manganese, zinc and cobalt acting as cofactors of the metalloproteins involved in sugar metabolism and/or glycosylation, the paper will outline the pathologies resulting from a dysregulation of these metals homeostasis and more particularly Congenital Disorders of Glycosylation (CDGs) caused by ion transporter defects. Highlighting of CDGs due to defects in SLC39A8 (ZIP8) and TMEM165, two proteins transporting manganese from the extracellular space to cytosol and from cytosol to the Golgi lumen, respectively, has emphasized the importance of manganese homeostasis for glycosylation. Based on our current knowledge of TMEM165 structure and functions, this review will draw a picture of known and putative mechanisms regulating manganese homeostasis in the secretory pathway. • Panorama of biometals essential in glycosylation process. • Diseases resulting from disturbances in biometals homeostasis. • Link between Mn homeostasis and CDG. [ABSTRACT FROM AUTHOR]

The TMEM165 gene encodes for a multiple pass membrane protein localized in the Golgi that has been linked to congenital disorders of glycosylation. The TMEM165 protein is a putative ion transporter that regulates H + /Ca ++ /Mn ++ homeostasis and pH in the Golgi. Previously, we identified TMEM165 as a potential biomarker for breast carcinoma in a glycoproteomic study using late stage invasive ductal carcinoma tissues with patient- matched adjacent normal tissues. The TMEM165 protein was not detected in non-malignant matched breast tissues and was detected in invasive ductal breast carcinoma tissues by mass spectrometry. Our hypothesis is that the TMEM165 protein confers a growth advantage to breast cancer. In this preliminary study we have investigated the expression of TMEM165 in earlier stage invasive ductal carcinoma and ductal carcinoma in situ cases. We created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration, tumor growth, and tumor vascularization in vivo . Furthermore, we find that TMEM165 expression alters the glycosylation of breast cancer cells and these changes promote the invasion and growth of breast cancer by altering the expression levels of key glycoproteins involved in regulation of the epithelial to mesenchymal transition such as E-cadherin. These studies illustrate new potential functions for this Golgi membrane protein in the control of breast cancer growth and invasion., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn 2+ , with an extra reported function in Ca 2+ and/or H + transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca 2+ signaling in yeast, and Mn 2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles., Competing Interests: Conflict of interest: The authors declare no conflicts of interest., (Copyright: © 2020 Thines et al.)

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 Ca 2+ - and Mn 2+ -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 Ca 2+ and Mn 2+ transporters abrogates Ca 2+ - and Mn 2+ -induced growth defects, excessive Mn 2+ 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 Ca 2+ and Mn 2+ 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 Ca 2+ and Mn 2+ , 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., (© 2020 Stribny et al.)

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 Ca 2+ 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 Ca 2+ 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 Ca 2+ -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., (© 2017 John Wiley & Sons Ltd.)

TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn 2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn 2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn 2+ -sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn 2+ -induced degradation of TMEM165., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein that functions in manganese uptake into the lumen. Manganese is needed in the thylakoid lumen to build up the inorganic Mn 4 CaO 5 cluster, the catalytic center for water oxidation, and is hence indispensable for oxygen evolution. A recent study revealed that PAM71 is well conserved in plants and shares homology to GCR1 DEPENDENT TRANSLATION FACTOR1 (GDT1) and TRANSMEMBRANE PROTEIN 165 (TMEM165) in Saccharomyces cerevisiae and Homo sapiens, respectively. In most eukaryotes only single members of this family, designated "Uncharacterized Protein Family 0016" (UPF0016), are present; however, plant genomes contain genes for several UPF0016 proteins. In Arabidopsis thaliana, this protein family comprises 5 members, which mainly differ in their N-terminal regions. PAM71 and its closest homolog PAM71-HL possess chloroplast transit peptides at their N-terminus. Two of the remaining 3 members are derived from a segmental chromosomal duplication event and lack an N-terminal extension. Thus, plants have evolved UPF0016 members residing in various compartments of the cell, whereas in non-plant eukaryotes just a Golgi localization occurs. The identification of PAM71 as a candidate Mn 2+ transporter opens the question on the function of the remaining plant members. Here we resume briefly our current knowledge of UPF0016 members in Arabidopsis in comparison to their yeast and human UPF0016 members.

Plasma membrane Ca(2+)-ATPase 2 (PMCA2) knockout mice showed that ~60% of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca(2+)-ATPase's 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca(2+)/H(+) antiporter (TMEM165) is expressed sufficiently in the Golgi of lactating mammary tissue to be a relevant contributor to secretory pathway mammary calcium transport. TMEM165 shows marked expression on day one of lactation when compared to timepoints prepartum. At peak lactation TMEM165 expression was 25 times greater than that of early pregnancy. Forced cessation of lactation resulted in a rapid ~50% decline in TMEM165 expression at 24h of involution and TMEM165 expression declined 95% at 96 h involution. It is clear that the timing, magnitude of TMEM165 expression and its Golgi location supports a role for this Golgi Ca2(+)/H(+) antiporter as a contributor to mammary Golgi calcium transport needs, in addition to the better-characterized roles of SPCA1&2., (Published by Elsevier Inc.)