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Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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ERp57 does not affect Ca2+ oscillations mediated by SERCA 2a, nor SERCA 2b glycosylation motif mutants. (A) Confocal images of Ca2+ oscillations in oocytes overexpressing SERCA 2b (n = 31), SERCA 2b + ERp57 (n = 39), SERCA 2a (n = 25), and SERCA 2a + ERp57 (n = 30). The traces represent two independent experiments with 12–20 oocytes per group. (B) Images of Ca2+ oscillations in oocytes overexpressing SERCA 2b-N1036A (n = 34), SERCA 2b-N1036A + ERp57 (n = 15), SERCA 2b-S1038A (n = 37), and SERCA 2b-S1038A + ERp57 (n = 16). The traces represent two independent experiments with 8–19 oocytes per group. (C) Histograms plot period and t1/2 for experiments in A and B, respectively. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b with SERCA 2b + ERp57 oocytes. (D) Western blots of SERCA 2a, SERCA 2b, or its mutants, and ERp57 from lysates of experimental oocytes as labeled. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Arrowheads indicate the protein as labeled at the top of each gel Bars: (A and B) 100 μm.
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fig7: ERp57 does not affect Ca2+ oscillations mediated by SERCA 2a, nor SERCA 2b glycosylation motif mutants. (A) Confocal images of Ca2+ oscillations in oocytes overexpressing SERCA 2b (n = 31), SERCA 2b + ERp57 (n = 39), SERCA 2a (n = 25), and SERCA 2a + ERp57 (n = 30). The traces represent two independent experiments with 12–20 oocytes per group. (B) Images of Ca2+ oscillations in oocytes overexpressing SERCA 2b-N1036A (n = 34), SERCA 2b-N1036A + ERp57 (n = 15), SERCA 2b-S1038A (n = 37), and SERCA 2b-S1038A + ERp57 (n = 16). The traces represent two independent experiments with 8–19 oocytes per group. (C) Histograms plot period and t1/2 for experiments in A and B, respectively. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b with SERCA 2b + ERp57 oocytes. (D) Western blots of SERCA 2a, SERCA 2b, or its mutants, and ERp57 from lysates of experimental oocytes as labeled. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Arrowheads indicate the protein as labeled at the top of each gel Bars: (A and B) 100 μm.

Mentions: CRT and CLNX inhibit Ca2+ oscillations via an interaction with the COOH-terminal sequence of SERCA 2b where a glycosylation consensus motif exists (John et al., 1998; Roderick et al., 2000). Furthermore, CRT does not interact with SERCA 2a, which lacks the 11th transmembrane segment and a COOH terminus in the ER lumen (John et al., 1998). ERp57 binds to CRT's P domain (Zapun et al., 1998; Oliver et al., 1999; Frickel et al., 2002; Leach et al., 2002). Based on these observations, we hypothesized that CRT binds the COOH-terminal sequence of the pump and recruits ERp57 to modulate L4 thiol groups and consequently pump activity. Because SERCA 2a also has the L4, we tested whether ERp57 modulates SERCA 2a directly or whether the effects are specific to SERCA 2b via CRT. We coexpressed ERp57 with SERCA 2a and found that ERp57 does not modulate Ca2+ oscillations in oocytes overexpressing this pump isoform (Fig. 7 A). We also coexpressed ERp57 with two mutants that eliminated the consensus glycosylation motif in SERCA 2b (SERCA 2b-N1036A and SERCA 2b-S1038A). We find that ERp57 does not modulate Ca2+ oscillations mediated by these two pump mutants (Fig. 7 B). Histograms of period and t1/2 are shown in Fig. 7 C, whereas Western blots for these experiments are shown in Fig. 7 D. Together, these experiments demonstrate that CRT is required to recruit ERp57 to the L4.


Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

ERp57 does not affect Ca2+ oscillations mediated by SERCA 2a, nor SERCA 2b glycosylation motif mutants. (A) Confocal images of Ca2+ oscillations in oocytes overexpressing SERCA 2b (n = 31), SERCA 2b + ERp57 (n = 39), SERCA 2a (n = 25), and SERCA 2a + ERp57 (n = 30). The traces represent two independent experiments with 12–20 oocytes per group. (B) Images of Ca2+ oscillations in oocytes overexpressing SERCA 2b-N1036A (n = 34), SERCA 2b-N1036A + ERp57 (n = 15), SERCA 2b-S1038A (n = 37), and SERCA 2b-S1038A + ERp57 (n = 16). The traces represent two independent experiments with 8–19 oocytes per group. (C) Histograms plot period and t1/2 for experiments in A and B, respectively. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b with SERCA 2b + ERp57 oocytes. (D) Western blots of SERCA 2a, SERCA 2b, or its mutants, and ERp57 from lysates of experimental oocytes as labeled. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Arrowheads indicate the protein as labeled at the top of each gel Bars: (A and B) 100 μm.
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fig7: ERp57 does not affect Ca2+ oscillations mediated by SERCA 2a, nor SERCA 2b glycosylation motif mutants. (A) Confocal images of Ca2+ oscillations in oocytes overexpressing SERCA 2b (n = 31), SERCA 2b + ERp57 (n = 39), SERCA 2a (n = 25), and SERCA 2a + ERp57 (n = 30). The traces represent two independent experiments with 12–20 oocytes per group. (B) Images of Ca2+ oscillations in oocytes overexpressing SERCA 2b-N1036A (n = 34), SERCA 2b-N1036A + ERp57 (n = 15), SERCA 2b-S1038A (n = 37), and SERCA 2b-S1038A + ERp57 (n = 16). The traces represent two independent experiments with 8–19 oocytes per group. (C) Histograms plot period and t1/2 for experiments in A and B, respectively. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b with SERCA 2b + ERp57 oocytes. (D) Western blots of SERCA 2a, SERCA 2b, or its mutants, and ERp57 from lysates of experimental oocytes as labeled. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Arrowheads indicate the protein as labeled at the top of each gel Bars: (A and B) 100 μm.
Mentions: CRT and CLNX inhibit Ca2+ oscillations via an interaction with the COOH-terminal sequence of SERCA 2b where a glycosylation consensus motif exists (John et al., 1998; Roderick et al., 2000). Furthermore, CRT does not interact with SERCA 2a, which lacks the 11th transmembrane segment and a COOH terminus in the ER lumen (John et al., 1998). ERp57 binds to CRT's P domain (Zapun et al., 1998; Oliver et al., 1999; Frickel et al., 2002; Leach et al., 2002). Based on these observations, we hypothesized that CRT binds the COOH-terminal sequence of the pump and recruits ERp57 to modulate L4 thiol groups and consequently pump activity. Because SERCA 2a also has the L4, we tested whether ERp57 modulates SERCA 2a directly or whether the effects are specific to SERCA 2b via CRT. We coexpressed ERp57 with SERCA 2a and found that ERp57 does not modulate Ca2+ oscillations in oocytes overexpressing this pump isoform (Fig. 7 A). We also coexpressed ERp57 with two mutants that eliminated the consensus glycosylation motif in SERCA 2b (SERCA 2b-N1036A and SERCA 2b-S1038A). We find that ERp57 does not modulate Ca2+ oscillations mediated by these two pump mutants (Fig. 7 B). Histograms of period and t1/2 are shown in Fig. 7 C, whereas Western blots for these experiments are shown in Fig. 7 D. Together, these experiments demonstrate that CRT is required to recruit ERp57 to the L4.

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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