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Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins.

Gatti G, Trifari S, Mesaeli N, Parker JM, Michalak M, Meldolesi J - J. Cell Biol. (2001)

Bottom Line: Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal.Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER.A model is proposed to explain this new process, that might also be valid for other luminal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Milan, 20129 Milan, Italy.

ABSTRACT
Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.

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Model for the CSQ oligomerization process. (A) MolScript (PDB1a8y) and sphere representation of the three domains of CSQ: red, domain 1 (residues 12–124); green, domain 2 (residues 125–228); blue, domain 3 (residues 229–352). The residues 326–333, which were not defined in the x-ray structure (Wang et al., 1998), are built manually using the Biopolymer option in InsightII software. (B) Representation of CSQ dimers. The NH2- and COOH-terminal tails are colored red and blue, respectively. The front-to-front dimer is shown at left, the back-to-back dimer at right, and the front-to-back dimer at center. (C) Regular repeating oligomers of CSQ. The oligomer shown at left is the front-to-front and back-to-back form revealed by x-ray structure (Wang et al., 1998). The oligomer shown at right is the front-to-back repeating form. In both oligomer forms, domain 2 has the same regular repeating spiral orientation. In contrast, the orientation of domains 1 and 3 differs in the two oligomeric forms. In the x-ray revealed oligomer, parallel spirals composed by alternating domain 1 followed by domain 3 are seen. In the potential oligomer, spirals of domain 1 and 3 run parallel to each other.
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fig7: Model for the CSQ oligomerization process. (A) MolScript (PDB1a8y) and sphere representation of the three domains of CSQ: red, domain 1 (residues 12–124); green, domain 2 (residues 125–228); blue, domain 3 (residues 229–352). The residues 326–333, which were not defined in the x-ray structure (Wang et al., 1998), are built manually using the Biopolymer option in InsightII software. (B) Representation of CSQ dimers. The NH2- and COOH-terminal tails are colored red and blue, respectively. The front-to-front dimer is shown at left, the back-to-back dimer at right, and the front-to-back dimer at center. (C) Regular repeating oligomers of CSQ. The oligomer shown at left is the front-to-front and back-to-back form revealed by x-ray structure (Wang et al., 1998). The oligomer shown at right is the front-to-back repeating form. In both oligomer forms, domain 2 has the same regular repeating spiral orientation. In contrast, the orientation of domains 1 and 3 differs in the two oligomeric forms. In the x-ray revealed oligomer, parallel spirals composed by alternating domain 1 followed by domain 3 are seen. In the potential oligomer, spirals of domain 1 and 3 run parallel to each other.

Mentions: Based on the specific information reported so far, on previous biochemical results (Cala and Jones, 1983; Maguire et al., 1997; Zhang et al., 1997) and the known crystal structure (Wang et al., 1998), we have developed a model for CSQ condensation using the Biopolymer option in InsightII software (Fig. 7) . The x-ray results had revealed that the protein may form “front-to-front” and “back-to-back” dimers (Fig. 7, F-F and B-B, respectively; Wang et al., 1998) in which the NH2 terminus and the COOH terminus could either fit into a groove between domains 1 and 3 or face away from the dimer interface, thereby enabling them to interact with other CSQ molecules. From these data, models of possible higher oligomers were developed. Only two regularly repeating structures showing the expected NH2- and COOH-terminal alignment were identified. The first is the front-to front and back-to-back oligomer revealed also by x-ray (Wang et al., 1998), the other is a “front-to-back” structure (Fig. 7, potential) that emerged from the analysis of the present data. The localization near the COOH terminus of the α-helical region including the critical residues, D341, D344, and D345, far away from the NH2-terminal sequence, appears compatible with its direct involvement in the CSQ–CSQ intermolecular binding. The polymers established according to the above models most likely correspond to the CSQ filaments revealed by electron microscopy within deep-etched SR terminal cisternae (Franzini-Armstrong et al., 1987).


Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins.

Gatti G, Trifari S, Mesaeli N, Parker JM, Michalak M, Meldolesi J - J. Cell Biol. (2001)

Model for the CSQ oligomerization process. (A) MolScript (PDB1a8y) and sphere representation of the three domains of CSQ: red, domain 1 (residues 12–124); green, domain 2 (residues 125–228); blue, domain 3 (residues 229–352). The residues 326–333, which were not defined in the x-ray structure (Wang et al., 1998), are built manually using the Biopolymer option in InsightII software. (B) Representation of CSQ dimers. The NH2- and COOH-terminal tails are colored red and blue, respectively. The front-to-front dimer is shown at left, the back-to-back dimer at right, and the front-to-back dimer at center. (C) Regular repeating oligomers of CSQ. The oligomer shown at left is the front-to-front and back-to-back form revealed by x-ray structure (Wang et al., 1998). The oligomer shown at right is the front-to-back repeating form. In both oligomer forms, domain 2 has the same regular repeating spiral orientation. In contrast, the orientation of domains 1 and 3 differs in the two oligomeric forms. In the x-ray revealed oligomer, parallel spirals composed by alternating domain 1 followed by domain 3 are seen. In the potential oligomer, spirals of domain 1 and 3 run parallel to each other.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196414&req=5

fig7: Model for the CSQ oligomerization process. (A) MolScript (PDB1a8y) and sphere representation of the three domains of CSQ: red, domain 1 (residues 12–124); green, domain 2 (residues 125–228); blue, domain 3 (residues 229–352). The residues 326–333, which were not defined in the x-ray structure (Wang et al., 1998), are built manually using the Biopolymer option in InsightII software. (B) Representation of CSQ dimers. The NH2- and COOH-terminal tails are colored red and blue, respectively. The front-to-front dimer is shown at left, the back-to-back dimer at right, and the front-to-back dimer at center. (C) Regular repeating oligomers of CSQ. The oligomer shown at left is the front-to-front and back-to-back form revealed by x-ray structure (Wang et al., 1998). The oligomer shown at right is the front-to-back repeating form. In both oligomer forms, domain 2 has the same regular repeating spiral orientation. In contrast, the orientation of domains 1 and 3 differs in the two oligomeric forms. In the x-ray revealed oligomer, parallel spirals composed by alternating domain 1 followed by domain 3 are seen. In the potential oligomer, spirals of domain 1 and 3 run parallel to each other.
Mentions: Based on the specific information reported so far, on previous biochemical results (Cala and Jones, 1983; Maguire et al., 1997; Zhang et al., 1997) and the known crystal structure (Wang et al., 1998), we have developed a model for CSQ condensation using the Biopolymer option in InsightII software (Fig. 7) . The x-ray results had revealed that the protein may form “front-to-front” and “back-to-back” dimers (Fig. 7, F-F and B-B, respectively; Wang et al., 1998) in which the NH2 terminus and the COOH terminus could either fit into a groove between domains 1 and 3 or face away from the dimer interface, thereby enabling them to interact with other CSQ molecules. From these data, models of possible higher oligomers were developed. Only two regularly repeating structures showing the expected NH2- and COOH-terminal alignment were identified. The first is the front-to front and back-to-back oligomer revealed also by x-ray (Wang et al., 1998), the other is a “front-to-back” structure (Fig. 7, potential) that emerged from the analysis of the present data. The localization near the COOH terminus of the α-helical region including the critical residues, D341, D344, and D345, far away from the NH2-terminal sequence, appears compatible with its direct involvement in the CSQ–CSQ intermolecular binding. The polymers established according to the above models most likely correspond to the CSQ filaments revealed by electron microscopy within deep-etched SR terminal cisternae (Franzini-Armstrong et al., 1987).

Bottom Line: Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal.Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER.A model is proposed to explain this new process, that might also be valid for other luminal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Milan, 20129 Milan, Italy.

ABSTRACT
Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.

Show MeSH
Related in: MedlinePlus