The Ca/H antiporter TMEM165 expression,localization in the developing,lactating and involuting mammary gland parallels the secretory pathway Ca ATPase (SPCA1)

Plasma membrane Ca²⁺-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 Ca²⁺-ATPase’s 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca²⁺/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 24 h 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.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy

TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn²⁺. Supplementation of cell with Mn²⁺ rescue the elongation process, confirming a role of TMEM165 in Mn²⁺ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFβ and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn²⁺ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.Subject terms: Glycobiology, Diseases     

C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca²⁺ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca²⁺ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca²⁺ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca²⁺ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.     

ACA12 is a deregulated isoform of plasma membrane Ca2+-ATPase of Arabidopsis thaliana

Plant auto-inhibited Ca²⁺-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca²⁺-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues—highly conserved in other ACA isoforms—localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.     

Efficient ER Exit and Vacuole Targeting of Yeast Sna2p Require Two Tyrosine‐Based Sorting Motifs

SNA (Sensitive to Na⁺) proteins form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members: Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C‐terminal region. Each of these is involved in a different Golgi‐to‐vacuole targeting pathway: the tyrosine 65 motif is involved in adaptor protein (AP‐1)‐dependent targeting, whereas the tyrosine 75 motif is involved in AP‐3‐dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were shown to be functional at two different steps of the secretory pathway, ER exit and Golgi‐to‐vacuole transport.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

Vacuolar Ca(2+) uptake

Calcium transporters that mediate the removal of Ca²⁺ from the cytosol and into internal stores provide a critical role in regulating Ca²⁺ signals following stimulus induction and in preventing calcium toxicity. The vacuole is a major calcium store in many organisms, particularly plants and fungi. Two main pathways facilitate the accumulation of Ca²⁺ into vacuoles, Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers. Here I review the biochemical and regulatory features of these transporters that have been characterised in yeast and plants. These Ca²⁺ transport mechanisms are compared with those being identified from other vacuolated organisms including algae and protozoa. Studies suggest that Ca²⁺ uptake into vacuoles and other related acidic Ca²⁺ stores occurs by conserved mechanisms which developed early in evolution.     

Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca²⁺-activated ion channel and Ca²⁺-activated phospholipid scramblase activities, requiring a high intracellular Ca²⁺ concentration (e.g., half-maximal effective Ca²⁺ concentration [EC₅₀] of [Ca²⁺]_i > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca²⁺-activated chloride channel exhibiting higher Ca²⁺ sensitivity (EC₅₀ of 1 μM) than ANO6, suggested that a homologous Ca²⁺-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca²⁺ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca²⁺-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6-1-6 showed higher sensitivity to Ca²⁺ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca²⁺ interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca²⁺-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca²⁺ sensitivity, implying direct interactions of Ca²⁺ with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca²⁺ sensitivity between ANO1 and ANO6.     

Bestrophin and TMEM16—Ca activated Cl channels with different functions

In the past, a number of candidates have been proposed to form Ca²⁺ activated Cl⁻ currents, but it is only recently that two families of proteins, the bestrophins and the TMEM16-proteins, recapitulate reliably the properties of Ca²⁺ activated Cl⁻ currents. Bestrophin 1 is strongly expressed in the retinal pigment epithelium, but also at lower levels in other cell types. Bestrophin 1 may form Ca²⁺ activated chloride channels and, at the same time, affect intracellular Ca²⁺ signaling. In epithelial cells, bestrophin 1 probably controls receptor mediated Ca²⁺ signaling. It may do so by facilitating Ca²⁺ release from the endoplasmic reticulum, thereby indirectly activating membrane localized Ca²⁺-dependent Cl⁻ channels. In contrast to bestrophin 1, the Ca²⁺ activated Cl⁻ channel TMEM16A (anoctamin 1, ANO1) shows most of the biophysical and pharmacological properties that have been attributed to Ca²⁺-dependent Cl⁻ channels in various tissues. TMEM16A is broadly expressed in both mouse and human tissues and is of particular importance in epithelial cells. Thus exocrine gland secretion as well as electrolyte transport by both respiratory and intestinal epithelia requires TMEM16A. Because of its role for Ca²⁺-dependent Cl⁻ secretion in human airways, it is likely to become a prime target for the therapy of cystic fibrosis lung disease, caused by defective cAMP-dependent Cl⁻ secretion. It will be very exciting to learn, how TMEM16A and other TMEM16-proteins are activated upon increase in intracellular Ca²⁺, and whether the other nine members of the TMEM16 family also form Cl⁻ channels with properties similar to TMEM16A.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

Yeast calmodulin known to be ubiquitylated in vivo in a Ca²⁺ dependent manner has long remained an orphan substrate. Here we identify Saccharomyces cerevisiae Asr1p as an ubiquitin E3 ligase for yeast calmodulin, a protein involved in calcium signaling. A short region within Asr1p-C harboring two putative calmodulin-binding motifs is sufficient and necessary for interaction with calmodulin. The interaction is direct, occurs in vivo and depends on physiological concentrations of Ca²⁺. A minimal set of purified proteins including Asr1p E3 ligase was sufficient for in vitro ubiquitylation of calmodulin, a reaction that required a functional Asr1p Ring domain. We propose a role of the Asr1p E3 ligase activity in coping with stress.     

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.     

Conformational plasticity of the calcium‐binding pocket in the Burkholderia glumae lipase: Remodeling induced by mutation of calcium coordinating residues

Most bacterial lipases bind one or more Ca²⁺ atoms at different locations and are a suitable case of study for investigating structural effects related to calcium binding, depletion, or mutation of calcium‐binding sites. Generally Ca²⁺ in microbial lipases can play a crucial role in the stabilization of the whole three‐dimensional structure by mediating long‐range effects. It has been recently demonstrated that calcium binding influences thermal stability of Burkholderia glumae lipase (BGL) through the restriction of conformational plasticity of specific regions. Moreover, calcium depletion results in a highly cooperative protein unfolding, eliciting protein aggregation. To further shed light on molecular mechanisms and structural features connected to calcium binding in microbial lipases, we present a molecular dynamics investigation, based on multiple‐replica approach at different temperatures, of BGL mutants targeting the calcium‐binding site. It turns out that additional acidic residues, which are conserved in other microbial lipases, help in overcoming effects induced by mutation of D241 Ca²⁺‐coordinating residue, upon rearrangements induced in the calcium binding site. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 117–126, 2011.     

Molecular underpinning of intracellular pH regulation on TMEM16F

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pH_i). We found that low pH_i attenuates, whereas high pH_i potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca²⁺ ([Ca²⁺]_i). We further demonstrate that TMEM16F pH_i sensitivity depends on [Ca²⁺]_i and exhibits a bell-shaped relationship with [Ca²⁺]_i: TMEM16F channel activation becomes increasingly pH_i sensitive from resting [Ca²⁺]_i to micromolar [Ca²⁺]_i, but when [Ca²⁺]_i increases beyond 15 µM, pH_i sensitivity gradually diminishes. The mutation of a Ca²⁺-binding residue that markedly reduces TMEM16F Ca²⁺ sensitivity (E667Q) maintains the bell-shaped relationship between pH_i sensitivity and Ca²⁺ but causes a dramatic shift of the peak [Ca²⁺]_i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pH_i regulatory effects on TMEM16F stem from the competition between Ca²⁺ and protons for the primary Ca²⁺-binding residues in the pore. Within the physiological [Ca²⁺]_i range, the protonation state of the primary Ca²⁺-binding sites influences Ca²⁺ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pH_i and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pH_i, including cancer.     

The C‐terminal calcium‐sensitive disordered motifs regulate isoform‐specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca²⁺‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca²⁺‐mediated folding suggesting that these disordered motifs are Ca²⁺‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca²⁺‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca²⁺‐dependent polymerization properties of CASQ. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 15–22, 2015.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility

Transmembrane protein 16E (TMEM16E) belongs to the TMEM16 family of proteins that have 10 transmembrane regions and appears to localize intracellularly. Although TMEM16E mutations cause bone fragility and muscular dystrophy in humans, its biochemical function is unknown. In the TMEM16 family, TMEM16A and -16B serve as Ca²⁺-dependent Cl⁻ channels, while TMEM16C, -16D, -16F, -16G, and -16J support Ca²⁺-dependent phospholipid scrambling. Here, we show that TMEM16E carries a segment composed of 35 amino acids homologous to the scrambling domain in TMEM16F. When the corresponding segment of TMEM16A was replaced by this 35-amino-acid segment of TMEM16E, the chimeric molecule localized to the plasma membrane and supported Ca²⁺-dependent scrambling. We next established TMEM16E-deficient mice, which appeared to have normal skeletal muscle. However, fertility was decreased in the males. We found that TMEM16E was expressed in germ cells in early spermatogenesis and thereafter and localized to sperm tail. TMEM16E^−/− sperm showed no apparent defect in morphology, beating, mitochondrial function, capacitation, or binding to zona pellucida. However, they showed reduced motility and inefficient fertilization of cumulus-free but zona-intact eggs in vitro. Our results suggest that TMEM16E may function as a phospholipid scramblase at inner membranes and that its defect affects sperm motility.