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Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

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Overexpression of GFP-SERCA2 fusion constructs reveals functional differences in SERCA2a and SERCA2b. (a)  Cartoon view of the fusion constructs used to tag SERCA isoforms at the NH2 terminus with the S65T increased fluorescence  mutant of GFP. (b) Western blot analysis demonstrates overexpression of GFP-SERCA2 fusion products detected by probing  the membrane with a polyclonal anti-SERCA2 antibody that was  raised in rabbit against a recombinant His-tagged fragment encompassing most of the cytoplasmic loop between M4 and M5  from rat SERCA2 (N1, gift of J. Lytton). Wild-type SERCA2a  and SERCA2b (lanes 1 and 3, respectively) migrated ∼27 kD below fusion protein products contained in extracts from oocytes  overexpressing GFP-SERCA2a and GFP-SERCA2b (lanes 2 and  4, respectively). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 5. Molecular size markers (in  kD) are indicated on the left (Kaleidoscope; Bio-Rad Laboratories). (c) High magnification confocal images of GFP fluorescence in oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b fusion proteins (left and middle, respectively). Note  that fluorescence is confined to the ER corridors between yolk  platelets. A control oocyte overexpressing cytosolic GFP is  shown (right). In this oocyte, fluorescence is more diffuse. Images  are 52 μm × 36 μm, and were acquired with an OZ confocal laser  scanning microscope (Noran Instruments, Middleton, WI). The  oocytes were excited at 488 nm and imaged with a 60× water immersion objective. Bar, ∼10 μm.
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Figure 2: Overexpression of GFP-SERCA2 fusion constructs reveals functional differences in SERCA2a and SERCA2b. (a) Cartoon view of the fusion constructs used to tag SERCA isoforms at the NH2 terminus with the S65T increased fluorescence mutant of GFP. (b) Western blot analysis demonstrates overexpression of GFP-SERCA2 fusion products detected by probing the membrane with a polyclonal anti-SERCA2 antibody that was raised in rabbit against a recombinant His-tagged fragment encompassing most of the cytoplasmic loop between M4 and M5 from rat SERCA2 (N1, gift of J. Lytton). Wild-type SERCA2a and SERCA2b (lanes 1 and 3, respectively) migrated ∼27 kD below fusion protein products contained in extracts from oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b (lanes 2 and 4, respectively). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 5. Molecular size markers (in kD) are indicated on the left (Kaleidoscope; Bio-Rad Laboratories). (c) High magnification confocal images of GFP fluorescence in oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b fusion proteins (left and middle, respectively). Note that fluorescence is confined to the ER corridors between yolk platelets. A control oocyte overexpressing cytosolic GFP is shown (right). In this oocyte, fluorescence is more diffuse. Images are 52 μm × 36 μm, and were acquired with an OZ confocal laser scanning microscope (Noran Instruments, Middleton, WI). The oocytes were excited at 488 nm and imaged with a 60× water immersion objective. Bar, ∼10 μm.

Mentions: Oocyte extracts used in Western blots were prepared from pools of 10 oocytes as previously described (Camacho and Lechleiter, 1995). The final pellet of each extract was resuspended in 50 μl of 1% SDS per oocyte equivalent, and was stored frozen in aliquots of one oocyte equivalent each. One oocyte equivalent of each fraction was loaded on an SDS gel, stained with Coomassie blue, and scanned on a UMAX Powerlook II scanner. Two invariant adjacent protein bands of ∼40 kD that appear in each extract were used as densitometric standards. The average of all densitometric readings was used to normalize the sample volume to load on SDS PAGE gels. To detect the ΔC mutant, samples were run on a 12% gel, and to detect the SERCA2 and GFP-tagged SERCA2 proteins, the samples were run on 8% gels by SDS-PAGE. To visualize the SERCA antigen, the membranes were probed with the polyclonal rabbit anti-SERCA antibody (C-4 Ab in Fig. 1 c and N1 Ab in Figs. 2 b and 7 a; both antibodies were a gift from J. Lytton, University of Calgary Health Sciences Centre, Department of Biochemistry and Molecular Biology, Calgary, Alberta, Canada). To detect the ΔC mutant of calreticulin (see Figs. 5 b and 6 d), oocyte fractions were probed with a primary rabbit anti-KDEL Ab that recognizes the COOH-terminal six amino acids of calreticulin (gift of M. Michalak, University of Alberta, Department of Biochemistry, Edmonton, Alberta, Canada). Note that this mutant contains the last six amino acids of calreticulin, including the KDEL ER retention signal, and thus it can be detected with this antibody (Camacho and Lechleiter, 1995). Alkaline phosphatase–conjugated secondary antibodies were used in all Western blots (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and colorimetric detection was accomplished by NBT/ BCIP (NitroBlue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate; Promega Corp., Madison, WI).


Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

Overexpression of GFP-SERCA2 fusion constructs reveals functional differences in SERCA2a and SERCA2b. (a)  Cartoon view of the fusion constructs used to tag SERCA isoforms at the NH2 terminus with the S65T increased fluorescence  mutant of GFP. (b) Western blot analysis demonstrates overexpression of GFP-SERCA2 fusion products detected by probing  the membrane with a polyclonal anti-SERCA2 antibody that was  raised in rabbit against a recombinant His-tagged fragment encompassing most of the cytoplasmic loop between M4 and M5  from rat SERCA2 (N1, gift of J. Lytton). Wild-type SERCA2a  and SERCA2b (lanes 1 and 3, respectively) migrated ∼27 kD below fusion protein products contained in extracts from oocytes  overexpressing GFP-SERCA2a and GFP-SERCA2b (lanes 2 and  4, respectively). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 5. Molecular size markers (in  kD) are indicated on the left (Kaleidoscope; Bio-Rad Laboratories). (c) High magnification confocal images of GFP fluorescence in oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b fusion proteins (left and middle, respectively). Note  that fluorescence is confined to the ER corridors between yolk  platelets. A control oocyte overexpressing cytosolic GFP is  shown (right). In this oocyte, fluorescence is more diffuse. Images  are 52 μm × 36 μm, and were acquired with an OZ confocal laser  scanning microscope (Noran Instruments, Middleton, WI). The  oocytes were excited at 488 nm and imaged with a 60× water immersion objective. Bar, ∼10 μm.
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Related In: Results  -  Collection

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Figure 2: Overexpression of GFP-SERCA2 fusion constructs reveals functional differences in SERCA2a and SERCA2b. (a) Cartoon view of the fusion constructs used to tag SERCA isoforms at the NH2 terminus with the S65T increased fluorescence mutant of GFP. (b) Western blot analysis demonstrates overexpression of GFP-SERCA2 fusion products detected by probing the membrane with a polyclonal anti-SERCA2 antibody that was raised in rabbit against a recombinant His-tagged fragment encompassing most of the cytoplasmic loop between M4 and M5 from rat SERCA2 (N1, gift of J. Lytton). Wild-type SERCA2a and SERCA2b (lanes 1 and 3, respectively) migrated ∼27 kD below fusion protein products contained in extracts from oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b (lanes 2 and 4, respectively). Oocyte extracts from control oocytes (H2O replacing mRNA) were run on lane 5. Molecular size markers (in kD) are indicated on the left (Kaleidoscope; Bio-Rad Laboratories). (c) High magnification confocal images of GFP fluorescence in oocytes overexpressing GFP-SERCA2a and GFP-SERCA2b fusion proteins (left and middle, respectively). Note that fluorescence is confined to the ER corridors between yolk platelets. A control oocyte overexpressing cytosolic GFP is shown (right). In this oocyte, fluorescence is more diffuse. Images are 52 μm × 36 μm, and were acquired with an OZ confocal laser scanning microscope (Noran Instruments, Middleton, WI). The oocytes were excited at 488 nm and imaged with a 60× water immersion objective. Bar, ∼10 μm.
Mentions: Oocyte extracts used in Western blots were prepared from pools of 10 oocytes as previously described (Camacho and Lechleiter, 1995). The final pellet of each extract was resuspended in 50 μl of 1% SDS per oocyte equivalent, and was stored frozen in aliquots of one oocyte equivalent each. One oocyte equivalent of each fraction was loaded on an SDS gel, stained with Coomassie blue, and scanned on a UMAX Powerlook II scanner. Two invariant adjacent protein bands of ∼40 kD that appear in each extract were used as densitometric standards. The average of all densitometric readings was used to normalize the sample volume to load on SDS PAGE gels. To detect the ΔC mutant, samples were run on a 12% gel, and to detect the SERCA2 and GFP-tagged SERCA2 proteins, the samples were run on 8% gels by SDS-PAGE. To visualize the SERCA antigen, the membranes were probed with the polyclonal rabbit anti-SERCA antibody (C-4 Ab in Fig. 1 c and N1 Ab in Figs. 2 b and 7 a; both antibodies were a gift from J. Lytton, University of Calgary Health Sciences Centre, Department of Biochemistry and Molecular Biology, Calgary, Alberta, Canada). To detect the ΔC mutant of calreticulin (see Figs. 5 b and 6 d), oocyte fractions were probed with a primary rabbit anti-KDEL Ab that recognizes the COOH-terminal six amino acids of calreticulin (gift of M. Michalak, University of Alberta, Department of Biochemistry, Edmonton, Alberta, Canada). Note that this mutant contains the last six amino acids of calreticulin, including the KDEL ER retention signal, and thus it can be detected with this antibody (Camacho and Lechleiter, 1995). Alkaline phosphatase–conjugated secondary antibodies were used in all Western blots (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and colorimetric detection was accomplished by NBT/ BCIP (NitroBlue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate; Promega Corp., Madison, WI).

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

Show MeSH
Related in: MedlinePlus