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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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L4 cysteine–deficient mutants of SERCA 2b exhibit higher frequencies of Ca2+ oscillations. (A) Western blots of SERCA 2b and L4 mutants were loaded as follows: (lane 1) SERCA 2b, (lane 2) SERCA 2b-C1SC2S and (lane 3) SERCA 2b-C1AC2A. Two oocyte equivalents were loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent four independent Western blots. (B) Confocal images of Ca2+ oscillations are shown in oocytes expressing SERCA 2b (n = 34) or the L4 cysteine–deficient mutants SERCA 2b-C1SC2AS (n = 40) and SERCA 2b-C1AC2A (n = 38). Traces represent two independent experiments with 15–20 oocytes per group. Histogram plots t1/2 for (1) SERCA 2b, (2) SERCA 2b-C1SC2S, and (3) SERCA 2b-C1AC2A expressing oocytes. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and each mutant group. Bar, 100 μm.
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fig2: L4 cysteine–deficient mutants of SERCA 2b exhibit higher frequencies of Ca2+ oscillations. (A) Western blots of SERCA 2b and L4 mutants were loaded as follows: (lane 1) SERCA 2b, (lane 2) SERCA 2b-C1SC2S and (lane 3) SERCA 2b-C1AC2A. Two oocyte equivalents were loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent four independent Western blots. (B) Confocal images of Ca2+ oscillations are shown in oocytes expressing SERCA 2b (n = 34) or the L4 cysteine–deficient mutants SERCA 2b-C1SC2AS (n = 40) and SERCA 2b-C1AC2A (n = 38). Traces represent two independent experiments with 15–20 oocytes per group. Histogram plots t1/2 for (1) SERCA 2b, (2) SERCA 2b-C1SC2S, and (3) SERCA 2b-C1AC2A expressing oocytes. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and each mutant group. Bar, 100 μm.

Mentions: To test whether the thiol groups in L4 are important in modulating Ca2+ oscillations enhanced by SERCA 2b, we imaged Ca2+ activity in oocytes overexpressing either the oxidizable form of SERCA 2b or two L4 cysteine–deficient mutants (SERCA 2b-C1SC2S and SERCA 2b-C1AC2A) that cannot form a disulfide bond and are a constitutively reduced form. Because these mutants did not express as efficiently as SERCA 2b in oocytes, we lowered expression levels of the pump to match expression levels of the mutants (Fig. 2 A). Under these conditions, we found that both mutants exhibited a higher frequency of Ca2+ oscillations and more rapid cytosolic Ca2+ uptake (i.e., shorter t1/2) than SERCA 2b (Fig. 2 B). We also overexpressed a single missense mutation found in a Darier disease pedigree that involves a cysteine to glycine mutation in L4 (SERCA 2b-C875G; Ruiz-Perez et al., 1999; Sakuntabhai et al., 1999; Ahn et al., 2003). When overexpressed in the oocytes, this mutant also exhibited a higher frequency of Ca2+ oscillations than the wild-type SERCA 2b (unpublished data). Together, with the previous data, our results suggest that ERp57 promotes disulphide bond formation in L4, thereby reducing the activity of the Ca2+ pump. When the disulfide bridge in L4 is disrupted, the Ca2+ ATPase appears to exhibit higher pump activity.


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

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

L4 cysteine–deficient mutants of SERCA 2b exhibit higher frequencies of Ca2+ oscillations. (A) Western blots of SERCA 2b and L4 mutants were loaded as follows: (lane 1) SERCA 2b, (lane 2) SERCA 2b-C1SC2S and (lane 3) SERCA 2b-C1AC2A. Two oocyte equivalents were loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent four independent Western blots. (B) Confocal images of Ca2+ oscillations are shown in oocytes expressing SERCA 2b (n = 34) or the L4 cysteine–deficient mutants SERCA 2b-C1SC2AS (n = 40) and SERCA 2b-C1AC2A (n = 38). Traces represent two independent experiments with 15–20 oocytes per group. Histogram plots t1/2 for (1) SERCA 2b, (2) SERCA 2b-C1SC2S, and (3) SERCA 2b-C1AC2A expressing oocytes. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and each mutant group. Bar, 100 μm.
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fig2: L4 cysteine–deficient mutants of SERCA 2b exhibit higher frequencies of Ca2+ oscillations. (A) Western blots of SERCA 2b and L4 mutants were loaded as follows: (lane 1) SERCA 2b, (lane 2) SERCA 2b-C1SC2S and (lane 3) SERCA 2b-C1AC2A. Two oocyte equivalents were loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent four independent Western blots. (B) Confocal images of Ca2+ oscillations are shown in oocytes expressing SERCA 2b (n = 34) or the L4 cysteine–deficient mutants SERCA 2b-C1SC2AS (n = 40) and SERCA 2b-C1AC2A (n = 38). Traces represent two independent experiments with 15–20 oocytes per group. Histogram plots t1/2 for (1) SERCA 2b, (2) SERCA 2b-C1SC2S, and (3) SERCA 2b-C1AC2A expressing oocytes. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and each mutant group. Bar, 100 μm.
Mentions: To test whether the thiol groups in L4 are important in modulating Ca2+ oscillations enhanced by SERCA 2b, we imaged Ca2+ activity in oocytes overexpressing either the oxidizable form of SERCA 2b or two L4 cysteine–deficient mutants (SERCA 2b-C1SC2S and SERCA 2b-C1AC2A) that cannot form a disulfide bond and are a constitutively reduced form. Because these mutants did not express as efficiently as SERCA 2b in oocytes, we lowered expression levels of the pump to match expression levels of the mutants (Fig. 2 A). Under these conditions, we found that both mutants exhibited a higher frequency of Ca2+ oscillations and more rapid cytosolic Ca2+ uptake (i.e., shorter t1/2) than SERCA 2b (Fig. 2 B). We also overexpressed a single missense mutation found in a Darier disease pedigree that involves a cysteine to glycine mutation in L4 (SERCA 2b-C875G; Ruiz-Perez et al., 1999; Sakuntabhai et al., 1999; Ahn et al., 2003). When overexpressed in the oocytes, this mutant also exhibited a higher frequency of Ca2+ oscillations than the wild-type SERCA 2b (unpublished data). Together, with the previous data, our results suggest that ERp57 promotes disulphide bond formation in L4, thereby reducing the activity of the Ca2+ pump. When the disulfide bridge in L4 is disrupted, the Ca2+ ATPase appears to exhibit higher pump activity.

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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