Diseases involving the Golgi calcium pump.

Secretory-pathway Ca2(+)-transport ATPases (SPCA) provide the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this organelle. Loss of one functional copy of the human SPCA1 gene (ATP2C1) causes Hailey-Hailey disease, a rare skin disorder characterized by recurrent blisters and erosions in the flexural areas. Here, we will review the properties and functional role of the SPCAs. The relationship between Hailey-Hailey disease and its defective gene (ATP2C1) will be adressed as well.

Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells.

The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals.

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments.

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.

The sarco-endoplasmic reticulum Ca2+ ATPase is required for development and muscle function in Caenorhabditis elegans.

The sarco-endoplasmic reticulum Ca(2+)-transport ATPase (SERCA) loads intracellular releasable Ca(2+) stores by transporting cytosolic Ca(2+) into the endoplasmic (ER) or sarcoplasmic reticulum (SR). We characterized the only SERCA homologue of the nematode Caenorhabditis elegans, which is encoded by the sca-1 gene. The sca-1 transcript is alternatively spliced in a similar mode as the vertebrate SERCA2 transcript, giving rise to two protein variants: CeSERCAa and CeSERCAb. These proteins showed structural and functional conservation to the vertebrate SERCA2a/b proteins. The CeSERCAs were primarily expressed in contractile tissues. Loss of CeSERCA through gene ablation or RNA interference resulted in contractile dysfunctioning and in early larval or embryonic lethality, respectively. Similar defects could be induced pharmacologically using the SERCA-specific inhibitor thapsigargin, which bound CeSERCA at a conserved site. The conservation of SERCA2 homologues in C. elegans will allow genetic and chemical suppressor analyses to identify promising drug targets and lead molecules for treatment of SERCA-related diseases such as heart disease.

Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica).

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.

Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA.

Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5'-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from -135 to -31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+.

Distribution and isoform diversity of the organellar Ca2+ pumps in the brain.

The gene family of organellar-type Ca2+ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca2+ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1-4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca2+, their possible roles in relation to Ca2+ stores in neurons are discussed.

cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 gene.

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5' region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3'-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5'-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177-6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

Purkinje neurons express the SERCA3 isoform of the organellar type Ca(2+)-transport ATPase.

We report the distribution of the sarco(endo)plasmic reticulum Ca2+ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca2+ pump has an approximately 5-fold lower affinity for Ca2+ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca(2+)-pump isoform could have important functional implications for the regulation of the cytosolic Ca2+ concentration in Purkinje neurons.

The SERCA3-type of organellar Ca2+ pumps.

Of all the SERCA pumps, SERCA3 was the latest to be described and the least well known. Its primary structure deviates more than usual from the other members of the SERCA family. It is not known whether its remarkably low affinity for Ca2+ (K0.5 > 1 microM) observed upon expression in the COS cell system occurs also in its normal cellular context. SERCA3 is particularly expressed at high levels in different types of blood cells and related cells like platelets, lymphocytes, mast cells and arterial endothelial cells. It is also found in cerebellar Purkinje neurons. The physiological significance of this expression pattern remains unknown.

A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.

An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.