ATP2C1 gene mutations in Hailey–Hailey disease and possible roles of SPCA1 isoforms in membrane trafficking

ATP2C1 gene codes for the secretory pathway Ca2+/Mn2+-ATPase pump type 1 (SPCA1) localizing at the golgi apparatus. Mutations on the human ATP2C1 gene, causing decreased levels of the SPCA1 expression, have been identified as the cause of the Hailey–Hailey disease, a rare skin disorder. In the last few years, several mutations have been described, and here we summarize how they are distributed along the gene and how missense mutations affect protein expression. SPCA1 is expressed in four different isoforms through alternative splicing of the ATP2C1 gene and none of these isoforms is differentially affected by any of these mutations. However, a better understanding of the tissue specific expression of the isoforms, their localization along the secretory pathway, their specific binding partners and the role of the C-terminal tail making isoforms different from each other, will be future goals of the research in this field.


Facts
Mutations occurring on the ATP2C1 gene clearly have no hotspots, although some mutations are redundant and the majority of missense mutations are spotted on specific exons. The C-terminal tails of two out of four SPCA1 isoforms display a sequence motif recognized by PDZ domains, potentially used to interact with different pools of protein and involved in different signaling pathways. SPCA1 has important roles in regulating membrane trafficking, not only as a Ca 2+ pump able to trigger the Ca 2+ influx into the lumen of the golgi apparatus (and of consequence the cytosolic peri-golgi Ca 2+ concentration/ signaling), but it also has a direct role in organizing cargo maturation/delivery from the golgi apparatus, which is imbalanced in cancer and other diseases.

Open Questions
Why mutations on the ATP2C1 gene cause a different etiology between human and mouse? Does the overlap of ATP2C1 gene with ASTE1 gene have a role in regulating the SPCA1 expression in a different manner between species?
Identification of proteins interacting with the C-terminal tails of the SPCA1 isoforms, and their possible role in mediating the function and the sub-organellar redistribution of the different SPCA1 isoforms in different cell types. Although SPCA1 is ubiquitously expressed in all the tissues, why mutations occurring on the ATP2C1 gene are mostly affecting the skin?
The study of intracellular membrane trafficking is important for the understanding of cellular structure and organelle function, and the coordinated cellular activities within complex organisms. The intracellular transport can be divided into different phases, which include the synthesis of lipids and proteins in the endoplasmic reticulum (ER), their folding and quality control, transport from ER-to-golgi apparatus and across the golgi apparatus, and delivery of cargoes to their final destinations. The golgi apparatus also participates in the posttranslational modifications (mostly glycosylation) of many proteins and lipids during their transport, and it is the central station of the intracellular secretory pathway. 1,2 The physiology of the secretory pathway and the golgi apparatus is finely regulated and maintained by pumps and channels that maintain the luminal pH/ion levels, making each sub-compartment of the golgi unique (i.e., the cis-side of the golgi apparatus is different from the trans-side). 3 The secretory pathway Ca 2+ -ATPase pump type 1 (SPCA1) regulates the golgi luminal Ca 2+ homeostasis, it is distributed along the secretory pathway membranes 4 and ubiquitously expressed in all tissues. 5 The human ATP2C1 gene encoding for SPCA1 is located on chromosome 3q21 and consists of 28 exons. 6,7 Alternative processing at the 3′-end of the human ATP2C1 pre-mRNA produces four distinct ATP2C1 splice variants (corresponding to SPCA1a-d proteins; Figure 1a), namely (i) SPCA1a from the splicing of exon 26 to exon 27 with the translation stop codon located in exon 27 producing a protein of 919 amino acids; (ii) SPCA1b which contains 939 amino acids and results from splicing of exons 27 to 28 following activation of the internal 5′-splice donor site D1; (iii) splicing of exons 26-28 gives rise to SPCA1c, which has 888 amino acids; (iv) splicing at internal site D2 in exon 27 to exon 28 gives rise to SPCA1d, which is the largest variant with 949 amino acids (Figure 1a′).
The resulting four SPCA1 protein isoforms differ in their C-terminal cytosolic tails and are organized in actuator domain (A), phosphorylation domain (P), nucleotide-binding domain (N), 5 stalk helices (S) in the cytoplasm, and 10 transmembrane helices (M) (Figure 1b). 8 The C-terminal tails of each SPCA1 isoforms, unique to each alternatively spliced product (Figure 1c), present characteristics for potential specific functions that we will discuss later. The alternative splicing is not present in other species where we have a single SPCA1 (Figure 1c).
A schematic representation of the ATP2C1 gene sequence and the relative encoded SPCA1 protein sequences is reported in Figure 2. Here, we show where the exons start/ finish (codons highlighted in yellow), which is the corresponding cytosolic (purple), transmembrane (gray) and luminal (blue) portion of SPCA1, as well as the C-terminal tail (azure).

Hailey-Hailey Disease
Mutations of the ATP2C1 gene have been reported in Hailey-Hailey disease (HHD) patients, 6,7 originally described by the Hailey brothers (Hugh Edward and William Howard) in 1939. 9 HHD is also known as familial benign chronic pemphigus 10 or familial benign pemphigus. 11 The prevalence of HHD is estimated to be 1:50 000. 12 HHD is a monoallelic genetic disorder inherited in an autosomal dominant pattern, 6,7 meaning that one copy of the altered gene is sufficient to cause the disorder. Comparison between genotype and phenotype failed to show a clear correlation between the nature of the mutation and the clinical features of HHD (age of onset, severity, progression). Extensive inter-familial and intrafamilial variation was noted in clinical features, as well as between families sharing the same mutation. 13 Mutations in the ATP2C1 gene reduce the amount of functional SPCA1. This abnormality impairs cells ability to store Ca 2+ normally. For unknown reasons, this abnormal Ca 2+ storage affects keratinocytes more than other cells types. The abnormal regulation of Ca 2+ impairs many cell functions, including cell adhesion. As a result, keratinocytes do not stick tightly to one another, which causes the epidermis to become fragile and less resistant to minor trauma. Because the skin is easily damaged, it develops raw, blistered areas, particularly in skin folds where there is moisture and friction.
Recent observations shed new light on a possible role for altered Ca 2+ homeostasis and responsiveness in HHD keratinocytes in creating an early defect in differentiation process due to a reduced production of involucrin. 14 Keratin expression was also delayed in acantholytic epidermal segments of HHD skin. 15 Because of decreased Ca 2+ stored in the lumen of the golgi apparatus of HHD keratinocytes, involucrin mRNA degraded in response to abnormally raised cytosolic Ca 2+ . 16 From dermatological perspectives, the HHD patients have been treated with topical steroid preparations to help outbreaks. Several drugs (antibiotics, antifungals, corticosteroids, etc.) keep under control the progression of the disease but are ineffective for severe chronic or relapsing forms. Some HHD patients found relief in laser resurfacing that burns off the top layer of the epidermis, allowing healthy nonaffected skin to regrow in its place.

Mutations on the Human ATP2C1 Gene
Here, we review the literature about the mutations occurring on the ATP2C1 gene, and give a comprehensive view of where these mutations fall along the gene sequence. Furthermore, we focus our attention on why some missense mutations affect SPCA1 efficiency without affecting the protein levels and address our interest to amino acidic residues that could probably have a role in the correct functioning of the protein and are not yet fully characterized. Finally, we focus our attention on the possible role of three different cytosolic tails of the SPCA1a, SPCA1b, and SPCA1d isoforms, and their potential for binding different partners.
Several mutations have been reported on the ATP2C1 gene in HHD patients, some of which were redundant even if no evident hotspots have been recognized so far. In Table 1 we summarize all the mutations reported so far, mentioning their localization along the gene sequence, the kind of mutation as well as the resultant change in the amino acid sequence. All the mutations generating truncated protein, which supposedly do not have a complete functionality or precise sub-organellar localization or are destabilized and therefore arguably destined for degradation, are indicated as premature termination codon (PTC).
After careful reviewing of literature using PubMed and the Chinese Biological Medicine Database (http://www.sinomed. ac.cn/zh/), we listed a total of 166 unique mutations on the ATP2C1 gene that have been reported in HHD to date. Of them,~55% lead to PTC, supporting the possibility that haploinsufficiency of ATP2C1 is a prevalent mechanism for the dominant inheritance of HHD. Twenty-four (~14%) were nonsense mutations, fifty-nine (~36%) were deletion/insertion mutations (of which five (~4%) are in-frame deletion and insertion mutations), thirty-four (~20%) were splice-site mutations, and forty-nine (~30%) were missense mutations (Table 1). Thus, many mutations predict the absence, or a marked reduction of the mutated ATP2C1 product via nonsense-mediated mRNA decay. Non-conservative amino acid changes in functional domains of the molecule are highly conserved between golgi Ca 2+ pumps from different species, and between other Ca 2+ -ATPases. In fact, some of these mutations occur at amino acid residues conserved between SPCA1 and SERCA1 (the ER ATPase Ca 2+ pump type 1); the latter has previously been studied by site-directed mutagenesis and the relevant mutations were shown to abrogate SERCA1 function. 17 Mutations are scattered along the ATP2C1 gene without apparent clustering, showing a substantial allelic heterogeneity, and are distributed all over the encoded sequence (Supplementary Figure 1), as well as in the intron splice sites generating alternative splicing and/or truncated proteins. Mutations affect all domains of the resulting protein. Rarely, mutations seem to ATG aag gtt gca cgt ttt caa aaa ata cct aat ggt gaa aat gag aca atg at t cct gta ttg aca tca aaa aaa gca agt gaa tta cca gtc agt gaa gtt gca ag c 19 109 att ctc caa gct gat ctt cag aat ggt cta aac aa a tgt gaa gtt agt cat ag g 37 163 cga gcc ttt cat ggc tgg aat aag ttt gat att agt gaa gat gag cca ctg tg g 55 217 aag aag tat att tct cag ttt aaa aat ccc ctt att atg ctg ctt ctg gct tc t 73 271 gca gtc atc agt gtt tta atg cat cag ttt gat gat gcc gtc agt atc act gt g 91 6 325 gca ata ctt atc gtt gtt aca gtt gcc ttt gtt cag gaa tat cgt tca gaa aaa 109 A 379 tct ctt gaa gaa ttg ag t aaa ctt gtg cca cca gaa tgc cat t gt gtg cgt gaa 127 433 gga aaa ttg gag cat aca ctt gcc cga gac ttg gt t cca ggt gat aca gtt tg c 145 487 ctt tct gtt ggg gat aga gtt cct gct gac tta cgc ttg ttt gag gct gtg gat 541 ctt tcc att gat gag tcc agc ttg aca ggt gag ac a acg cct tgt tct aag gt g 181 13 973 atg gtg aag aaa agg gcc att gtg aaa aag ctg cct att gtt gaa act ctg ggc 325 1027 tgc tgt aat gtg att tgt tca gat aaa act gga aca ctg acg aag aat gaa at g 343 1081 act gtt act cac ata tt t act tca gat ggt ctg cat gct gag gtt act gga gtt 361 1135 ggc tat aat caa ttt ggg gaa gtg att gtt gat ggt gat gtt gtt cat gga tt c 379 1189 tat aac cca gct gtt agc aga att gtt gag gcg ggc tgt gtg tgc aat gat gc t 397 Figure 2 Identification of transmembrane domains vs cytosolic or luminal domains of SPCA1. For simplicity, SPCA1a (NP_055197) has been represented; other isoforms have identical sequence until the C-terminal tail. The highlighted codons in green and in red are the starting and stop codon, respectively. Highlighted in yellow is the first codon of each corresponding numbered exon. Highlighted in gray is the transmembrane domain. The highlighted amino acids in purple are cytoplasmic, whereas those highlighted in blue are luminal. The highlighted amino acids in azure represent the cytosolic C-terminal tail (underlined is the KXD/E motif putative COPI-binding site). The bold typed red and blue amino acids are the residues directly involved in the binding of Ca 2+ /Mn 2+ and Mg 2+ , respectively. Of note, the putative COPI-binding motif KXD/E is just below the M10 domain. The prediction of these domains/residues was solved by similarity (UniProt database)  29,30 and 2395C4T causing R799X. 18,25,[31][32][33][34] The above single residues represent 'hotspots' for mutations, and notably, they all occur at CpG sites where the possibility of mutations is higher. 35 The fact that the majority of the mutations cause PTC through frameshift or single-base-pair substitution points to haploinsufficiency as the prevalent mechanism for the dominant inheritance of HHD. 6,7 However, the possibility that at least some mutations cause the disease through a dominant negative mechanism cannot be excluded. 13 Interestingly, only one mutation was found in the most 3′ region (exons 27-28) of the gene where differential splicing generates the four different transcripts. 22 The single reported 1243 gta att aga aac aat act cta atg ggg aag cca aca gaa ggg gcc tta att gc t 1297 ctt gca atg aag atg ggt ctt gat gga ctt caa caa gac tac atc aga aaa gc t 433 1351 gaa tac cct ttt agc tct gag caa aag tgg atg gct gtt aag tgt gta cac cg a 451 1405 aca cag cag gac aga cca gag att tgt ttt atg aa a ggt gct tac gaa caa gta 469 1459 att aag tac tgt act aca tac cag agc aaa ggg cag acc ttg aca ctt act ca g 487 1513 cag cag aga gat gtg tac caa caa gag aag gca cgc atg ggc tca gcg gga ct c 505 1567 aga gtt ctt gct ttg gct tct ggt cct gaa ctg gga cag ctg aca ttt ctt gg c 523 1621 ttg gtg gga atc att gat cca cct aga act ggt gtg aaa gaa gct gtt aca ac a 541 1675 ctc att gcc tca gga gta tca ata aaa atg att act gga gat tca cag gag ac t 1729 gca gtt gca atc gcc agt cgt ctg gga ttg tat tcc aaa act tcc cag tca gt c 577 1783 tca gga gaa gaa ata gat gca atg gat gtt cag cag ctt tca caa ata gta cc a 595 1837 aag gtt gca gta ttt tac aga gct agc cca agg cac aag atg aaa att att aag 1891 tcg cta cag aag aac ggt tca gtt gta gcc atg aca gga gat gga gta aat ga t 631 1945 gca gtt gct ctg aag gct gca gac att gga gtt gcg atg ggc cag act ggt ac a 649 1999 gat gtt tgc aaa gag gca gca gac atg atc cta gtg gat gat gat ttt caa ac c 667 2053 ata atg tct gca atc gaa gag ggt aaa ggg att tat aat aac att aaa aat tt c 685 2107 gtt aga ttc cag ctg agc acg agt ata gca gca tta act tta atc tca ttg gc t 703 2161 aca tta atg aac ttt cct aat cct ctc aat gcc atg cag att ttg tgg atc aa t 721 2215 att att atg gat gga ccc cca gct cag agc ctt gga gta gaa cca gtg gat aa a 739 2269 gat gtc att cgt aaa cct cct cgc aac tgg aaa gac agc att ttg act aaa aac 757 2323 ttg ata ctt aaa ata ctt gtt tca tca ata atc att gtt tgt ggg act ttg tt t 775 2377 gtc ttc tgg cgt gag cta cga gac aat gtg att aca cct cga gac aca aca at g 793 2431 acc ttc aca tgc ttt gtg ttt ttt gac atg ttc aat gca cta agt tcc aga tc c 811 . The mutation was found in a patient with a family history of HHD and suffering from classical HHD symptoms. 22 Notably, this mutation caused the loss of the C termini of the SPCA1a, SPCA1b and SPCA1d isoforms but not of SPCA1c that does not include exon 27. Thus, SPCA1c, the shortest of the isoforms, misses the M10, and therefore, its stability and consequent ability to function as a Ca 2+ pump is rather questionable. 36 Nevertheless, if SPCA1c was effectively expressed, we can conclude that it is not functional enough to overcome the loss of the other three isoforms at least in the skin of the patient presenting the mentioned S887X mutation. 22 Remarkably, this observation suggests that the mRNA encoding for SPCA1c is probably not translated as previously suggested. 37 Furthermore, the mutation on exon 27 causes a reduction in SPCA1a, SPCA1b and SPCA1d of 33, 52 and 63 amino acids, respectively, resulting in a partial truncation of the M10 and of the full cytoplasmic tail, similarly to what happens in SPCA1c (888 amino acids). Of consequence, this mutation is predicted to determine an incomplete expression and degradation of these SPCA1 isoforms thus resembling SPCA1c. Hereafter, we will no more consider SPCA1c in this discussion.
Splice variants differ in their C-terminal tail sequence ( Figure 1c) and this is likely to be important for the functionality of the pump. Indeed, the C-terminal tail could have a role in mediating interactions with cytoplasmic effectors for intracellular signaling or for targeting the single isoforms to specific sub-organellar localization. Presently, no cytoplasmic interacting proteins have been found for the SPCA1 C-terminal domain. However, specific interactions of different SPCA1 tails with proteins involved in membrane trafficking (i.e., golgi matrix, Ca 2+ -binding proteins, Arf/Rab family members, cytoskeleton, etc.) could support a specific role for C-terminal tail in the differential distribution of the protein at suborganellar level. This would in turn make them unique not only in triggering the Ca 2+ influx into the golgi apparatus, but also in mediating different cytoplasmic signaling, thus orchestrating membrane trafficking at different levels along the secretory pathway. Obviously, these speculations need to be experimentally proven.
No further mutations have been discovered in the 3′-end of the ATP2C1 gene. In other words, once the ATP2C1 gene is mutated (independently if missense, nonsense, insertion and/or duplication or part of the primary sequence) the functionality of all the isoforms is equally compromised in HHD patients, and at least in the skin, levels of SPCA1 appear to be not sufficient. However, up-regulation of SERCA2 (the ER ATPase Ca 2+ pump type 2) has been recently shown to (at least partly) compensate for the decreased levels of SPCA1 in HHD. 38 Therefore, although SPCA1 is ubiquitously expressed, is there differential expression of the isoforms? Is the compensatory role for non-functional or depleted SPCA1 by other pumps tissue specific? Is there a general mechanism? If so, how does it work? Even if some tentative explanations have been given, 38,39 these open questions need answers to fully understand the role and distribution of the SPCA1 isoforms.

Missense Mutations on the ATP2C1 Gene
Missense mutations generate single amino acid substitution, not leading to a PTC. A quick view on where the missense mutations occur along the ATP2C1 gene sequence gives an idea of their preferential distribution in few exons. In particular, almost two-thirds (63%) of the missense mutations (31/49) localize in only five exons (exon 12, 13, 18, 21, and 23; Table 1). These exons encode for M4, P, ATP and M5/M6 domains, respectively ( Figure 3). Amino acids located on M4, M5 and M6 (together with M8) transmembrane domains are critical for Ca 2+ /Mn 2+ binding. It is not surprising that affecting residues in these regions, either directly involved in Ca 2+ -binding or compromising the stability or structure of the protein, may cause a severe effect on SPCA1 functionality, also without a reduction in its levels. Looking at the conservation across species we found high level of homology of these exons and conservativeness of the resulting amino acid coded by the codons where missense mutations occur (Figure 3), confirming their critical role.
Several residues have been reported to have a critical and direct role in Ca 2+ /Mn 2+ binding, which are conserved throughout species 40 and also in SPCA2, the homolog of SPCA1. 41 Missense mutation D778A in the yeast homolog (PMR1) resulted in a loss of function mutant apparently defective for the transport of both Ca 2+ and Mn 2+ , whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn 2+ transport. 42 In a similar study, 26 2485 cag acc aag tct gtg ttt gag att gga ctc tgc agt aat aga atg ttt tgc ta t 829 2539 gca gtt ctt gga tcc atc atg gga caa tta cta gtt att tac ttt cct ccg ct t 847 2593 cag aag gtt ttt cag act gag agc cta agc ata ctg gat ctg ttg ttt ctt ttg 2647 ggt ctc acc tca tca gtg tgc ata gtg gca gaa att ata aag aag gtt gaa ag g 883 Human ATP2C1 gene mutations were summarized. The mutations are grouped for deletion/insertion (azure), nonsense (orange), missense (yellow), acceptor/donor splice (green). Nucleotides are reported in italic along the table as well as in the figure legend and along the full manuscript. We found some reported mutations were inaccurate or not unified. Therefore, we revised or collated some descriptions according to the reported cDNA reference sequence (GenBank accession No. NM_AF181120). 7 Appendix 1: A polymorphism was wrongly reported to be a new mutation 2323C4T generating Y711H. 87 After careful check with the correct reading frame this was not a mutation but a polymorphism. 88 Appendix 2: A resuming panel graphs the amount and relative percentage of each kind of mutations and PTC. Appendix 3: The 884-904delCCATTGTGGTCACAGTGACGC mutation and consequent amino acid 296-302delIVVTVTL was incorrectly reported, 18 while it referred to as a 21bp deletion located at 932-952 and amino acid 311-317delPIVVTVT. This mutation did not generate the reported missense mutation P295V; 18 Figure 3 Missense mutations mostly affect critical SPCA1 domains. Most of the missense mutations localize on exons 12, 13-16, 18, 21, and 23 of ATP2C1 gene coding for the indicated protein domains (M4, P, N, S5, M5-M6) crucial for the protein enzymatic activity and Ca 2+ /Mn 2+ binding. In human HHD patients the missense mutations cause the indicated amino acid changes, supporting a crucial role for them in functionality of the SPCA1. Of note, the mutations localize on highly conserved residue/codon between the considered species, highlighting the importance of those encoded amino acids. In blue are indicated the different amino acids compared to the human sequence, in red where the mutations occur. Alignments were obtained from Ensembl database as described in Figure 1 ATP2C1 mutations and SPCA1 isoforms M Micaroni et al G783 and V335 in PMR1 showed conformation-sensitive packing at the cytoplasmic interface, suggesting that these residues are important in organizing the entry gate for Mn 2+ ions. 43 Missense mutation can also cause a change in protein conformation affecting possible interaction of SPCA1 with other proteins, like cofilin, which has been found to be crucial in recruiting actin to mediate the cargo sorting that interact directly with SPCA1 nucleotide domain through an interaction mediated by Ca 2+ ions. 44 Remarkably, these examples highlight how a possible missense mutation can affect SPCA1 resulting not only in a less efficient pump, but also accounting for its diminished ability to interact with other proteins. In some HHD patients this scenario results in a severe etiology, comparable to the situation where mutations on ATP2C1 gene cause PTC, but in the presence of unaffected levels of total protein expression.

ATP2C1 Gene: Human Versus Murine
Another aspect to highlight is the different etiology occurring between humans and mice as a consequence of ATP2C1 mutations. In humans, HHD rarely degenerates in skin cancer although squamous cell carcinoma and basal cell carcinoma arising in lesions of HHD have been described in the literature. [45][46][47] However, which is the relationship, if any, between skin cancer development and HHD has not been deeply investigated. To our knowledge, there are no reports of melanoma or non-cutaneous malignant neoplasms associated with HHD.
On the other side, heterozygotic mice exhibit no evidence of skin lesions indicative of HHD, meanwhile generated skin cancer in adult. 48,49 Null mutant embryos exhibited growth retardation, failure of neural tube closure, but normal hematopoiesis and cardiovascular development, resulting in embryonic lethality. 48 At subcellular level, golgi membranes of Spca1 − / − embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased golgi biogenesis. 48 Increased golgi-associated vesicles and a marked reduction of ribosomes on rough ER were also observed. 48 Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. 48 However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipids by the golgi. 48 Possibly, reduction of the proper levels of Spca1 resulted in alteration in the activity of the glycosyl-transferases due to a decreased accumulation of Mn 2+ in the golgi lumen, which could contribute to the secretory pathway stress as well as apoptosis due to a decreased of Ca 2+ handling.
Recently, an important difference between human and murine genomes that could explain the differential outcome of mutations in the two species has been reported. 50 The Atp2c1 gene in mice expresses only one isoform of the gene, not presenting alternative splicing events at the 3′-end of the gene like the human counterpart. The last exon of the ATP2C1 human gene overlapped in the antisense direction with the last exon of the ASTE1 (also named HT001) gene, which codes for a protein of unknown function. 50 ATP2C1/ASTE1 overlap (not present in the murine genome) could have a role in regulating isoform expression, or more largely, the full SPCA1 expression. 50 This sort of regulation could have in turn a protective role that is imbalanced once one of the two alleles is mutated in humans, whereas not in mice. 50 Putative Interacting Domains in the C-terminal Cytosolic Tails of SPCA1 Isoforms As suggested above, different SPCA1 isoforms due to their unique C termini could have a different pattern of distribution and functional roles. The primary sequence of the C-terminal tail of each isoform is reported in Figure 1c. An intriguing possibility is that the uniqueness of their function is a consequence of the interaction with different protein partners. It is well known that many resident proteins display in their tails, motifs like sorting signals responsible for retrieval or retention. For instance, the C-terminal KKXX motif, identified in many ER-localized type I membrane proteins, can function as an ER-retention signal to retrieve ER-resident membrane proteins from the golgi via a direct interaction with coat protein complex I (COPI) coatomer. [51][52][53] Another example is the semiconserved (F/L)-(L/V)-(S/T) motif, which has been shown to function as a golgi-retention signal by interacting with COPI vesicles via Vps74p and thereby maintaining the steady golgi localization of glycosyl-transferases in yeast. 54 Recently, a conserved retention function and COPI-binding ability for the KXD/E motif has been shown in the golgi apparatus through the evolution in different species and in many endomembrane proteins. 55,56 Using bioinformatics tools (see Figure 4 legend), we analyzed the SPCA1 sequences in the areas corresponding to the terminal exons 26-28 in terms of secondary structure propensity (Figure 4a), predicted transmembrane helices topology (Figure 4b), and interaction with putative protein partners (Figure 4c). We found that the KXD/E motif is present in isoforms SPCA1a, SPCA1b, and SPCA1d, in the cytosolic tail immediately after the M10 (Figure 4c) in a region that is predicted to be a helical extension of M10 in all three isoforms (compare Figures 4a and b). This motif is also highly conserved throughout different species suggesting functional importance ( Figure 4d). Therefore, SPCA1 is potentially involved in the interaction with COPI. This reinforces the idea that SPCA1 could be involved in regulating local fusion/fission of cargo domain/ vesicles carrying proteins along the secretory pathway, triggering the cytosolic Ca 2+ level in the peri-golgi membranes, where Ca 2+ -binding proteins are temporarily recruited to organize and regulate the membrane fusion/fission, a fundamental process for cargo progression. 4,57 The analysis of the potential of C-terminal tail residues to interact with protein partners also highlighted additional sequences, which are differentially present in the four isoforms (Figure 4c). In particular, the isoforms SPCA1b and SPCA1d both end with the C-terminal sequence EDVSCV, which is predicted to be a protein interaction site (Figure 4c). By inspecting the literature we realized that this motif perfectly reproduces the structural features that are necessary for recognition by class I PDZ domains: (i) being at the C-terminal of a protein and (ii) matching the consensus for the last three residues S/T-X-Φ-COO − , 58 where X is any residue and Φ is a hydrophobic residue (often a valine). Thus, this motif is strongly predicted to interact with the PDZ domains of other proteins. 59 Several proteins containing the PDZ domain can potentially interact with the tail of SPCA1b and SPCA1d modulating the function of the pump. Interestingly, evidences that the C-terminal tail of SPCA1 could interact with the golgi matrix arise from a recent observation of the interaction of C-terminal valine-bearing cargoes moving through the golgi apparatus by binding to GRASP55 and GRASP65, two golgi matrix proteins that contain PDZ domains. 60 PDZ domain recognition of the C-terminal tail is involved in golgi maintenance and in cargo progression. 60,61 The SPCA1 isoforms present a valine residue as last amino acid residue in the C-terminal position (Figures  1c and 4c).
Also the FLEV motif present at the C-terminal end of SPCA1a and in the cytoplasmic tail of SPCA1d is predicted to be a protein binding motif (Figure 4c). Interestingly, this motif is also present in rat calsequestrin (CASQ; AAA75480). CASQ regulates Ca 2+ -binding in the sarcoplasmic reticulum in  Figure 4 Bioinformatics analysis of SPCA1 isoforms C-terminal region. (a) Secondary structure propensities in the proteins' regions encoded by terminal exons were predicted with the program PSIPRED. 92 Highlighted in pink is the corresponding sequences in the KXD/E motif. Predictions indicate that this motif is located in a helical extension spanning outside transmembrane helix M10 (see also b). Interestingly this helix is predicted to have different length in the three isoforms where it is present (a, b and d).
(b) Transmembrane topology prediction was performed using the algorithms MEMSAT3 (ref. 93 cardiac muscle cells. However, this domain is not conserved between the human and murine proteins. This unique feature of the human protein could favor specific protein interactions by SPCA1 isoforms to mediate signaling and trigger the modulation of Ca 2+ levels in the interested subcellular compartment. In the case of CASQ the interactors could be triadin and junctin. 62 Analogs could have the same role in the golgi apparatus for SPCA1, working as Ca 2+ -mediated sensing probes to regulate the pump activity. The above observations reinforce the hypothesis for a functional role of each different C-terminal tail in mediating interaction with different proteins.
Importance of SPCA1 in Membrane Trafficking: What is the Possible Role of each Isoform?
In addition to the maintenance of intra-golgi Ca 2+ and Mn 2+ , which has direct effects on Ca 2+ -dependent proteases 63 and Mn 2+ -dependent glycosyltransferases 64 (see also below), there is also evidence that SPCA1 may affect cytosolic Ca 2+ signaling. [65][66][67][68][69] Ca 2+ released from the golgi leads to a localized increase in cytosolic Ca 2+ that stimulates vesicle fusion, thus contributing to intra-golgi transport of cargo, 70 consistent with the distribution of SPCA1 in the golgi apparatus. 4 Thus, maintenance of luminal Ca 2+ stores by SPCA1 may enable Ca 2+ signaling events that directly affect the transport of cargocontaining domains from ER to the golgi apparatus and from there to their final destinations. 3 If so, a deficiency in this signaling function likely contributes to the mislocalization of proteins in response to a decreased SPCA1 activity/level. [71][72][73] Furthermore, effects on the ER were also observed in mammalian cells. Depletion of SPCA1 affected degradation of a mutant glycoproteins via ERAD, although it did not affect degradation of a non-glycoprotein substrates. 71 Signaling pathways that mediate ER stress responses were intact and functional; however, SPCA1-deficient cells were highly sensitive to treatment with tunicamycin or thapsigargin, both of which cause ER stress and activate ER stress responses. 71 Evidences of golgi stress, secondary effects on the ER, and stress responses were categorized as survival responses or apoptotic responses. 72 Although the role of SPCA1 in the Ca 2+ homeostasis of the golgi apparatus and its role in membrane trafficking has been well described and accepted, 70,73 the role of single isoforms still needs to be investigated. Possible differential distribution along the secretory pathway at sub-organelle level as well as at tissue level remains completely uninvestigated too. SPCA1 isoforms differ from each other in their C-terminal tails that is not directly involved in the regulation of Ca 2+ -binding. However, many Ca 2+ -binding proteins are recruited on the golgi membranes during the trafficking pulse (cPLA2, calmodulin, SNARE cofactors, etc.). 3 These proteins are recruited on the golgi membranes through a local release of Ca 2+ from the golgi apparatus and they should be released as soon as they fulfill their own function. 3 The recruited Ca 2+ -binding proteins are addressed to the so-called lateral rims (or non compact zones) of the golgi stacks and in both the cisand trans-golgi area where SPCA1 is clearly localized. 4 Whether SPCA1 C terminus tails are involved in establishing temporary contacts with Ca 2+ -binding recruited proteins remains unknown. SPCA1 is also able to transport Mn 2+ ions into the golgi with high affinity, an ability that SERCAs do not possess. As Mn 2+ is not used for signaling like Ca 2+ , the main reason for removing it from the cytosol is to prevent Mn 2+ toxicity. 74 Several proteins within the pathway require either Ca 2+ and Mn 2+ , or other divalent ions to function as metal cofactors, such as amino-peptidase P, 75 pro-protein convertases 76 and sulfotransferarese. 77 Even if there is no differential efficiency in the removal of ions by each SPCA1 isoform, potential differential sub-organellar localization of the isoforms may reflect the pump activity involved in different Ca 2+ /Mn 2+ -dependent pathways.
The inefficient removal of Ca 2+ /Mn 2+ from the cytosol by SPCA1, with consequent decreased accumulation in the golgi, causes alteration in intracellular signaling and posttranscriptional protein modifications. Mn 2+ is a fundamental cofactor of the golgi resident enzymes mannosidases. 78 Reduced removal of Ca 2+ from the cytosol during the arrival and the passage of cargo proteins to and through the golgi apparatus, significantly reduces physiological protein secretion. 4 Furthermore, SPCA1 seems to have a direct role in organizing the cargo sorting at the trans-golgi network 79,80 and subsequent departure. 81 Functional SPCA1 is important for insulin-like growth factor receptor (IGF1R) processing in basal-like breast cancer, and inhibition of SPCA1 'may offer an alternative strategy to direct inhibitors of IGF1R and attenuate the processing of other proprotein convertase substrates…'. 82 However, which of the SPCA1 isoforms, if any, works at this level still needs to be defined.

Membrane Trafficking in HHD Keratinocytes
In the skin of HHD patients, one of the altered clusters of proteins affected by non-functional or depleted SPCA1 are the adhesion proteins. The loss of one functional copy of the ATP2C1 gene in HHD is characterized by the development of skin lesions and an associated loss of cell-cell adhesion. 6,7 Proteins necessary for desmosome formation (as well as extracellular matrix components) are likely to be directly involved in cellular interactions and are affected in SPCA1 +/ − cells. 83 Desmosomes are molecular complexes of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular keratin cytoskeletal filaments. Desmoglein and desmocollin, two components of the desmosome, are members of the cadherin family of cell adhesion molecules. They are transmembrane proteins that bridge the space between adjacent epithelial cells by way of homophilic binding of their extracellular domains to other desmosomal cadherins on the adjacent cell. 83 Both have five extracellular domains and have Ca 2+ -binding motifs. Defects in anchoring to cytoskeletal constituents can also reflect possible intracellular morphological defects as observed in golgi apparatus. 84 Another important finding relating SPCA1 depletion and protein secretion in keratinocytes comes from a study on involucrin (a protein that makes up the cornified envelope of keratinocytes and is expressed in response to increased ATP2C1 mutations and SPCA1 isoforms M Micaroni et al cytosolic Ca 2+ ). 14 Involucrin is expressed at lower levels in cells from HHD patients or cells treated with SPCA1 RNAi when compared to normal keratinocytes. 14 Decreased level of involucrin was caused by lower involucrin mRNA levels in HHD keratinocytes; decreased involucrin mRNA, in turn, was caused by increased rates of involucrin mRNA degradation. 14 Ca 2+ -sensitive involucrin AP-1 promoter activity was increased, both in HHD keratinocytes and in RNAi experimental model, suggesting compensatory promoter up-regulation in the face of increased mRNA degradation. 14 Thus, transport/secretion of desmosomes components are impaired by SPCA1 depletion.
Further observations revealed that claudins are also regulated by SPCA1. 85 Claudins are a family of proteins that are the most important components of the tight junctions, establishing a barrier that controls the flow of molecules in the intercellular space between the cells of an epithelium. In SPCA1-depleted keratinocytes protein levels of components of desmosomes, adherens and tight junctions did not show marked changes except for claudin1 and 4 that were increased. 85 Whether and which SPCA1 isoforms are implicated in the regulation of specific cell adhesion protein trafficking is unknown. Nevertheless, the reason why desmosomes/tight junctions are affected more in the skin than in other epithelial tissues has to be investigated.

Conclusions
Distribution of SPCA1 isoforms along the secretory pathway in different cell types could lead to a better understanding of which isoform is doing what, when, and where. Considering that SPCA1 is ubiquitously expressed, the fundamental role of this protein in regulating the Ca 2+ /Mn 2+ physiology 86 and membrane trafficking in different cell types/tissues could be due to specific clusters of SPCA1 isoforms. Further investigations to clarify the role of each SPCA1 isoforms will benefit the understanding of the biology of secretion in health and disease, and will impact on several pathological conditions.