Limits...
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.

Show MeSH
The interaction between ERp57 and L4 is Ca2+ dependent and specific. (A) GST pull-down assays performed at the indicated [Ca2+] in micromolars. The gel is loaded as follows: input of in vitro–translated L4 (lane 1); pull downs for 30 μg GST control (lanes 2–5); pull downs for 10 μg GST-ERp57 (lanes 6–9). Proteins were resolved through 15% SDS-PAGE. L4 migrates ∼11 kD (109 aa). Data represent three independent experiments. Histogram depicts densitometric analysis from these experiments. L4 bands were normalized to the intensity of the band at 300 μM Ca2+. Asterisks indicate statistical significance (P < 0.05, one-way ANOVA). (B) The interaction between ERp57 and L4 is specific by GST pull-down assay. Lanes were loaded as follows: input of in vitro–translated L4 (lane 1); 30 μg GST negative control at the indicated [Ca2+] (lanes 2 and 3); 10 μg GST-ERp57 (lanes 4 and 5); 10 μg GST-PDI (lanes 6 and 7). Proteins were resolved through 15% SDS-PAGE. The gel represents three independent experiments. (Right gel) Coomassie blue stain of 3 μg purified GST (lane 1); 1 μg GST-ERp57 (lane 2), and 1 μg GST-PDI (lane 3). Proteins were resolved through 13% SDS-PAGE. GST migrates ∼27 kD, and GST-ERp57 and GST-PDI migrate ∼80 kD (mature rPDI is 489 aa, mature hERp57 is 481 aa). The arrowheads correspond to L4. (C) The L4 protein localizes to the ER. (Top) Coexpression in Xenopus oocytes of GFP-L4 (green) with DsRed-IP3R (red). The IP3R is used as a marker of ER localization. The overlay (yellow) demonstrates that the L4 localizes in the ER. (Bottom) Cytosolic GFP (green) used a negative control, is coexpressed with DsRed-IP3R (red). Note that the cytosolic GFP expression pattern is more diffused than that of the GFP-L4 and that there is no overlap of the two proteins indicating lack of colocalization. High magnification of the small square regions are shown as insets in each figure. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171954&req=5

fig5: The interaction between ERp57 and L4 is Ca2+ dependent and specific. (A) GST pull-down assays performed at the indicated [Ca2+] in micromolars. The gel is loaded as follows: input of in vitro–translated L4 (lane 1); pull downs for 30 μg GST control (lanes 2–5); pull downs for 10 μg GST-ERp57 (lanes 6–9). Proteins were resolved through 15% SDS-PAGE. L4 migrates ∼11 kD (109 aa). Data represent three independent experiments. Histogram depicts densitometric analysis from these experiments. L4 bands were normalized to the intensity of the band at 300 μM Ca2+. Asterisks indicate statistical significance (P < 0.05, one-way ANOVA). (B) The interaction between ERp57 and L4 is specific by GST pull-down assay. Lanes were loaded as follows: input of in vitro–translated L4 (lane 1); 30 μg GST negative control at the indicated [Ca2+] (lanes 2 and 3); 10 μg GST-ERp57 (lanes 4 and 5); 10 μg GST-PDI (lanes 6 and 7). Proteins were resolved through 15% SDS-PAGE. The gel represents three independent experiments. (Right gel) Coomassie blue stain of 3 μg purified GST (lane 1); 1 μg GST-ERp57 (lane 2), and 1 μg GST-PDI (lane 3). Proteins were resolved through 13% SDS-PAGE. GST migrates ∼27 kD, and GST-ERp57 and GST-PDI migrate ∼80 kD (mature rPDI is 489 aa, mature hERp57 is 481 aa). The arrowheads correspond to L4. (C) The L4 protein localizes to the ER. (Top) Coexpression in Xenopus oocytes of GFP-L4 (green) with DsRed-IP3R (red). The IP3R is used as a marker of ER localization. The overlay (yellow) demonstrates that the L4 localizes in the ER. (Bottom) Cytosolic GFP (green) used a negative control, is coexpressed with DsRed-IP3R (red). Note that the cytosolic GFP expression pattern is more diffused than that of the GFP-L4 and that there is no overlap of the two proteins indicating lack of colocalization. High magnification of the small square regions are shown as insets in each figure. Bar, 5 μm.

Mentions: To investigate the interaction between ERp57 and its potential target L4, we performed an in vitro GST pull-down experiment under various Ca2+ concentrations: the highest being 300 μM to mimic full Ca2+ store content and the lowest being 10 μM to mimic Ca2+ store depletion. Interestingly, ERp57 binds to L4 in a Ca2+-dependent manner: the interaction is strongest at high Ca2+ (300 μM) and is significantly reduced at low Ca2+ (10 μM; Fig. 5 A). To test the specificity of this interaction we performed a similar GST pull-down experiment with GST-PDI. Neither GST-PDI nor GST alone interacted with the L4 (Fig. 5 B). This suggests that the interaction between ERp57 and L4 is specific. Moreover, this interaction occurs preferentially at high Ca2+ concentrations that are indicative of full Ca2+ stores (Pozzan et al., 1994).


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

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

The interaction between ERp57 and L4 is Ca2+ dependent and specific. (A) GST pull-down assays performed at the indicated [Ca2+] in micromolars. The gel is loaded as follows: input of in vitro–translated L4 (lane 1); pull downs for 30 μg GST control (lanes 2–5); pull downs for 10 μg GST-ERp57 (lanes 6–9). Proteins were resolved through 15% SDS-PAGE. L4 migrates ∼11 kD (109 aa). Data represent three independent experiments. Histogram depicts densitometric analysis from these experiments. L4 bands were normalized to the intensity of the band at 300 μM Ca2+. Asterisks indicate statistical significance (P < 0.05, one-way ANOVA). (B) The interaction between ERp57 and L4 is specific by GST pull-down assay. Lanes were loaded as follows: input of in vitro–translated L4 (lane 1); 30 μg GST negative control at the indicated [Ca2+] (lanes 2 and 3); 10 μg GST-ERp57 (lanes 4 and 5); 10 μg GST-PDI (lanes 6 and 7). Proteins were resolved through 15% SDS-PAGE. The gel represents three independent experiments. (Right gel) Coomassie blue stain of 3 μg purified GST (lane 1); 1 μg GST-ERp57 (lane 2), and 1 μg GST-PDI (lane 3). Proteins were resolved through 13% SDS-PAGE. GST migrates ∼27 kD, and GST-ERp57 and GST-PDI migrate ∼80 kD (mature rPDI is 489 aa, mature hERp57 is 481 aa). The arrowheads correspond to L4. (C) The L4 protein localizes to the ER. (Top) Coexpression in Xenopus oocytes of GFP-L4 (green) with DsRed-IP3R (red). The IP3R is used as a marker of ER localization. The overlay (yellow) demonstrates that the L4 localizes in the ER. (Bottom) Cytosolic GFP (green) used a negative control, is coexpressed with DsRed-IP3R (red). Note that the cytosolic GFP expression pattern is more diffused than that of the GFP-L4 and that there is no overlap of the two proteins indicating lack of colocalization. High magnification of the small square regions are shown as insets in each figure. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171954&req=5

fig5: The interaction between ERp57 and L4 is Ca2+ dependent and specific. (A) GST pull-down assays performed at the indicated [Ca2+] in micromolars. The gel is loaded as follows: input of in vitro–translated L4 (lane 1); pull downs for 30 μg GST control (lanes 2–5); pull downs for 10 μg GST-ERp57 (lanes 6–9). Proteins were resolved through 15% SDS-PAGE. L4 migrates ∼11 kD (109 aa). Data represent three independent experiments. Histogram depicts densitometric analysis from these experiments. L4 bands were normalized to the intensity of the band at 300 μM Ca2+. Asterisks indicate statistical significance (P < 0.05, one-way ANOVA). (B) The interaction between ERp57 and L4 is specific by GST pull-down assay. Lanes were loaded as follows: input of in vitro–translated L4 (lane 1); 30 μg GST negative control at the indicated [Ca2+] (lanes 2 and 3); 10 μg GST-ERp57 (lanes 4 and 5); 10 μg GST-PDI (lanes 6 and 7). Proteins were resolved through 15% SDS-PAGE. The gel represents three independent experiments. (Right gel) Coomassie blue stain of 3 μg purified GST (lane 1); 1 μg GST-ERp57 (lane 2), and 1 μg GST-PDI (lane 3). Proteins were resolved through 13% SDS-PAGE. GST migrates ∼27 kD, and GST-ERp57 and GST-PDI migrate ∼80 kD (mature rPDI is 489 aa, mature hERp57 is 481 aa). The arrowheads correspond to L4. (C) The L4 protein localizes to the ER. (Top) Coexpression in Xenopus oocytes of GFP-L4 (green) with DsRed-IP3R (red). The IP3R is used as a marker of ER localization. The overlay (yellow) demonstrates that the L4 localizes in the ER. (Bottom) Cytosolic GFP (green) used a negative control, is coexpressed with DsRed-IP3R (red). Note that the cytosolic GFP expression pattern is more diffused than that of the GFP-L4 and that there is no overlap of the two proteins indicating lack of colocalization. High magnification of the small square regions are shown as insets in each figure. Bar, 5 μm.
Mentions: To investigate the interaction between ERp57 and its potential target L4, we performed an in vitro GST pull-down experiment under various Ca2+ concentrations: the highest being 300 μM to mimic full Ca2+ store content and the lowest being 10 μM to mimic Ca2+ store depletion. Interestingly, ERp57 binds to L4 in a Ca2+-dependent manner: the interaction is strongest at high Ca2+ (300 μM) and is significantly reduced at low Ca2+ (10 μM; Fig. 5 A). To test the specificity of this interaction we performed a similar GST pull-down experiment with GST-PDI. Neither GST-PDI nor GST alone interacted with the L4 (Fig. 5 B). This suggests that the interaction between ERp57 and L4 is specific. Moreover, this interaction occurs preferentially at high Ca2+ concentrations that are indicative of full Ca2+ stores (Pozzan et al., 1994).

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.

Show MeSH