Limits...
SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Show MeSH

Related in: MedlinePlus

SERCA1 cDNA clones isolated from normal liver and the predicted structure of encoded proteins. (A) cDNA structure of the SERCA1 adult isoform with a stop codon in exon 22 (SERCA1a) and of the neonatal isoform characterized by exon 22 splicing and a stop codon in exon 23 (SERCA1b). Spliced isoforms (S1T+4 and S1T−4) are characterized by exon 4 and/or exon 11 splicing. Exon 11 splicing leads to a frameshift, encoding a 22-aa peptide (▪), and a premature stop codon in exon 12. (B) Predicted topological structure of S1T proteins as compared with SERCA1. (Left) SERCA1. Numbers below drawings indicate transmembrane domains (M). Phosphorylatable Asp-351 is indicated. • and ♦ indicate transmembrane residues involved in calcium-binding sites I and II, respectively, and that bind calcium by a side-chain oxygen atom. ▴ corresponds to the Asp-800 residue that participates in both sites I and II. ⋄ correspond to residues involved in calcium-binding site II and that bind calcium by the backbone oxygen atom. ▪ indicate cytoplasmic calcium-binding residues. (Middle and right) S1T proteins. The 22-aa COOH-terminal peptide is depicted as a dotted line, and the peptide (35 aa) encoded by exon 4 and deleted in S1T−4 is in gray (right). (C) Three-dimensional structure of rabbit SERCA1a, according to Toyoshima et al. 2000, using Rasmol 2.7.1 and the 1EUL PDB file (http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1EUL, see supplementary materials for directions on viewing under different orientations). The A transduction (A), phosphorylation (P), and nucleotide-binding (N) domains are indicated. Secondary structures are displayed in ribbons. Those that are common to SERCA1 and S1T proteins are in color: β-sheets are in dark yellow, and cytosolic α helices are in magenta. Residues from Leu-396 to Gly-994 (COOH-terminal) removed by the splicing of exon 11 in S1T are in gray. Transmembrane α helices M1, M2, M3, and M4 are depicted in yellow, green, violet, and red, respectively. The peptide Val-74 to Gln-108 encoded by exon 4 and deleted in the S1T−4 protein is indicated in blue. Residues Phe-73 and Glu-109, which come in contact in S1T−4, are indicated. Phosphorylatable Asp-351 and calcium-binding residue Glu-309 are also shown. Calcium ions are space filled and colored in cyan. (D) Hypothetical three-dimensional structure of S1T deduced from that of rabbit SERCA1a. Residue Val-395 in S1T is followed by a 22-aa COOH-terminal peptide generated by the frameshift and depicted as a dotted line. Its structure is unknown. The region colored in blue is removed in the S1T−4 protein and presumably leads to an important structural rearrangement. The position of the calcium atom located near residue Glu-309 is hypothetical. Residues Glu-58 and Gln-108, which are close to Glu-309 and may participate in the channelling of calcium in S1T+4 dimers along the side of transmembrane domains M2–M4, are displayed in wireframe.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192035&req=5

Figure 1: SERCA1 cDNA clones isolated from normal liver and the predicted structure of encoded proteins. (A) cDNA structure of the SERCA1 adult isoform with a stop codon in exon 22 (SERCA1a) and of the neonatal isoform characterized by exon 22 splicing and a stop codon in exon 23 (SERCA1b). Spliced isoforms (S1T+4 and S1T−4) are characterized by exon 4 and/or exon 11 splicing. Exon 11 splicing leads to a frameshift, encoding a 22-aa peptide (▪), and a premature stop codon in exon 12. (B) Predicted topological structure of S1T proteins as compared with SERCA1. (Left) SERCA1. Numbers below drawings indicate transmembrane domains (M). Phosphorylatable Asp-351 is indicated. • and ♦ indicate transmembrane residues involved in calcium-binding sites I and II, respectively, and that bind calcium by a side-chain oxygen atom. ▴ corresponds to the Asp-800 residue that participates in both sites I and II. ⋄ correspond to residues involved in calcium-binding site II and that bind calcium by the backbone oxygen atom. ▪ indicate cytoplasmic calcium-binding residues. (Middle and right) S1T proteins. The 22-aa COOH-terminal peptide is depicted as a dotted line, and the peptide (35 aa) encoded by exon 4 and deleted in S1T−4 is in gray (right). (C) Three-dimensional structure of rabbit SERCA1a, according to Toyoshima et al. 2000, using Rasmol 2.7.1 and the 1EUL PDB file (http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1EUL, see supplementary materials for directions on viewing under different orientations). The A transduction (A), phosphorylation (P), and nucleotide-binding (N) domains are indicated. Secondary structures are displayed in ribbons. Those that are common to SERCA1 and S1T proteins are in color: β-sheets are in dark yellow, and cytosolic α helices are in magenta. Residues from Leu-396 to Gly-994 (COOH-terminal) removed by the splicing of exon 11 in S1T are in gray. Transmembrane α helices M1, M2, M3, and M4 are depicted in yellow, green, violet, and red, respectively. The peptide Val-74 to Gln-108 encoded by exon 4 and deleted in the S1T−4 protein is indicated in blue. Residues Phe-73 and Glu-109, which come in contact in S1T−4, are indicated. Phosphorylatable Asp-351 and calcium-binding residue Glu-309 are also shown. Calcium ions are space filled and colored in cyan. (D) Hypothetical three-dimensional structure of S1T deduced from that of rabbit SERCA1a. Residue Val-395 in S1T is followed by a 22-aa COOH-terminal peptide generated by the frameshift and depicted as a dotted line. Its structure is unknown. The region colored in blue is removed in the S1T−4 protein and presumably leads to an important structural rearrangement. The position of the calcium atom located near residue Glu-309 is hypothetical. Residues Glu-58 and Gln-108, which are close to Glu-309 and may participate in the channelling of calcium in S1T+4 dimers along the side of transmembrane domains M2–M4, are displayed in wireframe.

Mentions: We cloned SERCA1 transcripts from normal liver and obtained 25 clones. Upon analysis with full-length SERCA1, SERCA1 exon 11, and SERCA1 exon 4 specific probes, 17 clones corresponded to SERCA1 and 8 clones were found to be characterized by exon 11 splicing (S1T+4), including two that also exhibited exon 4 splicing (S1T−4). This result was confirmed by sequence analysis. Exon 11 splicing leads to a frameshift encoding 22 aa (PKVSMRRSARPPRQHSPPWWRR) followed by a premature stop codon in exon 12 (Fig. 1 A).


SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

SERCA1 cDNA clones isolated from normal liver and the predicted structure of encoded proteins. (A) cDNA structure of the SERCA1 adult isoform with a stop codon in exon 22 (SERCA1a) and of the neonatal isoform characterized by exon 22 splicing and a stop codon in exon 23 (SERCA1b). Spliced isoforms (S1T+4 and S1T−4) are characterized by exon 4 and/or exon 11 splicing. Exon 11 splicing leads to a frameshift, encoding a 22-aa peptide (▪), and a premature stop codon in exon 12. (B) Predicted topological structure of S1T proteins as compared with SERCA1. (Left) SERCA1. Numbers below drawings indicate transmembrane domains (M). Phosphorylatable Asp-351 is indicated. • and ♦ indicate transmembrane residues involved in calcium-binding sites I and II, respectively, and that bind calcium by a side-chain oxygen atom. ▴ corresponds to the Asp-800 residue that participates in both sites I and II. ⋄ correspond to residues involved in calcium-binding site II and that bind calcium by the backbone oxygen atom. ▪ indicate cytoplasmic calcium-binding residues. (Middle and right) S1T proteins. The 22-aa COOH-terminal peptide is depicted as a dotted line, and the peptide (35 aa) encoded by exon 4 and deleted in S1T−4 is in gray (right). (C) Three-dimensional structure of rabbit SERCA1a, according to Toyoshima et al. 2000, using Rasmol 2.7.1 and the 1EUL PDB file (http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1EUL, see supplementary materials for directions on viewing under different orientations). The A transduction (A), phosphorylation (P), and nucleotide-binding (N) domains are indicated. Secondary structures are displayed in ribbons. Those that are common to SERCA1 and S1T proteins are in color: β-sheets are in dark yellow, and cytosolic α helices are in magenta. Residues from Leu-396 to Gly-994 (COOH-terminal) removed by the splicing of exon 11 in S1T are in gray. Transmembrane α helices M1, M2, M3, and M4 are depicted in yellow, green, violet, and red, respectively. The peptide Val-74 to Gln-108 encoded by exon 4 and deleted in the S1T−4 protein is indicated in blue. Residues Phe-73 and Glu-109, which come in contact in S1T−4, are indicated. Phosphorylatable Asp-351 and calcium-binding residue Glu-309 are also shown. Calcium ions are space filled and colored in cyan. (D) Hypothetical three-dimensional structure of S1T deduced from that of rabbit SERCA1a. Residue Val-395 in S1T is followed by a 22-aa COOH-terminal peptide generated by the frameshift and depicted as a dotted line. Its structure is unknown. The region colored in blue is removed in the S1T−4 protein and presumably leads to an important structural rearrangement. The position of the calcium atom located near residue Glu-309 is hypothetical. Residues Glu-58 and Gln-108, which are close to Glu-309 and may participate in the channelling of calcium in S1T+4 dimers along the side of transmembrane domains M2–M4, are displayed in wireframe.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192035&req=5

Figure 1: SERCA1 cDNA clones isolated from normal liver and the predicted structure of encoded proteins. (A) cDNA structure of the SERCA1 adult isoform with a stop codon in exon 22 (SERCA1a) and of the neonatal isoform characterized by exon 22 splicing and a stop codon in exon 23 (SERCA1b). Spliced isoforms (S1T+4 and S1T−4) are characterized by exon 4 and/or exon 11 splicing. Exon 11 splicing leads to a frameshift, encoding a 22-aa peptide (▪), and a premature stop codon in exon 12. (B) Predicted topological structure of S1T proteins as compared with SERCA1. (Left) SERCA1. Numbers below drawings indicate transmembrane domains (M). Phosphorylatable Asp-351 is indicated. • and ♦ indicate transmembrane residues involved in calcium-binding sites I and II, respectively, and that bind calcium by a side-chain oxygen atom. ▴ corresponds to the Asp-800 residue that participates in both sites I and II. ⋄ correspond to residues involved in calcium-binding site II and that bind calcium by the backbone oxygen atom. ▪ indicate cytoplasmic calcium-binding residues. (Middle and right) S1T proteins. The 22-aa COOH-terminal peptide is depicted as a dotted line, and the peptide (35 aa) encoded by exon 4 and deleted in S1T−4 is in gray (right). (C) Three-dimensional structure of rabbit SERCA1a, according to Toyoshima et al. 2000, using Rasmol 2.7.1 and the 1EUL PDB file (http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1EUL, see supplementary materials for directions on viewing under different orientations). The A transduction (A), phosphorylation (P), and nucleotide-binding (N) domains are indicated. Secondary structures are displayed in ribbons. Those that are common to SERCA1 and S1T proteins are in color: β-sheets are in dark yellow, and cytosolic α helices are in magenta. Residues from Leu-396 to Gly-994 (COOH-terminal) removed by the splicing of exon 11 in S1T are in gray. Transmembrane α helices M1, M2, M3, and M4 are depicted in yellow, green, violet, and red, respectively. The peptide Val-74 to Gln-108 encoded by exon 4 and deleted in the S1T−4 protein is indicated in blue. Residues Phe-73 and Glu-109, which come in contact in S1T−4, are indicated. Phosphorylatable Asp-351 and calcium-binding residue Glu-309 are also shown. Calcium ions are space filled and colored in cyan. (D) Hypothetical three-dimensional structure of S1T deduced from that of rabbit SERCA1a. Residue Val-395 in S1T is followed by a 22-aa COOH-terminal peptide generated by the frameshift and depicted as a dotted line. Its structure is unknown. The region colored in blue is removed in the S1T−4 protein and presumably leads to an important structural rearrangement. The position of the calcium atom located near residue Glu-309 is hypothetical. Residues Glu-58 and Gln-108, which are close to Glu-309 and may participate in the channelling of calcium in S1T+4 dimers along the side of transmembrane domains M2–M4, are displayed in wireframe.
Mentions: We cloned SERCA1 transcripts from normal liver and obtained 25 clones. Upon analysis with full-length SERCA1, SERCA1 exon 11, and SERCA1 exon 4 specific probes, 17 clones corresponded to SERCA1 and 8 clones were found to be characterized by exon 11 splicing (S1T+4), including two that also exhibited exon 4 splicing (S1T−4). This result was confirmed by sequence analysis. Exon 11 splicing leads to a frameshift encoding 22 aa (PKVSMRRSARPPRQHSPPWWRR) followed by a premature stop codon in exon 12 (Fig. 1 A).

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Show MeSH
Related in: MedlinePlus