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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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Point mutations in the CSQ B binding site. (A) Graphic representation of the point mutations in two distinct, three acidic amino acid sets within site B. (B, B′, C, and C′) L6 cells were cotransfected with one point-mutated construct together with HA-CRT. The figure compares the immunofluorescence patterns in the cells labeled with anti-CSQ (B and C) and anti-HA (B′ and C′) Abs, as indicated at the top of the panels.
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fig4: Point mutations in the CSQ B binding site. (A) Graphic representation of the point mutations in two distinct, three acidic amino acid sets within site B. (B, B′, C, and C′) L6 cells were cotransfected with one point-mutated construct together with HA-CRT. The figure compares the immunofluorescence patterns in the cells labeled with anti-CSQ (B and C) and anti-HA (B′ and C′) Abs, as indicated at the top of the panels.

Mentions: To identify the two binding sites at higher resolution, experiments were set up in which the full-length protein was deleted of short amino acid sequences, or point mutated at strategically located sites. When either site A or site B was deleted, the distribution of the mutants was diffuse (Fig. 3, B and C) , coinciding largely with that of CRT (Fig. 3, B′ and C′). Largely diffuse distribution was also obtained after site-specific mutation of full-length CSQ (Fig. 4 A) at three acidic amino acids in site B, D341A, E344A, and D345A (Fig. 4 C). In contrast, site-specific mutation of three, more proximal glutamic acid residues (E337A, E338A, and E340A) failed to affect the punctate distribution, which remained unchanged with respect to intact CSQ (Fig. 4 B).


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)

Point mutations in the CSQ B binding site. (A) Graphic representation of the point mutations in two distinct, three acidic amino acid sets within site B. (B, B′, C, and C′) L6 cells were cotransfected with one point-mutated construct together with HA-CRT. The figure compares the immunofluorescence patterns in the cells labeled with anti-CSQ (B and C) and anti-HA (B′ and C′) Abs, as indicated at the top of the panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196414&req=5

fig4: Point mutations in the CSQ B binding site. (A) Graphic representation of the point mutations in two distinct, three acidic amino acid sets within site B. (B, B′, C, and C′) L6 cells were cotransfected with one point-mutated construct together with HA-CRT. The figure compares the immunofluorescence patterns in the cells labeled with anti-CSQ (B and C) and anti-HA (B′ and C′) Abs, as indicated at the top of the panels.
Mentions: To identify the two binding sites at higher resolution, experiments were set up in which the full-length protein was deleted of short amino acid sequences, or point mutated at strategically located sites. When either site A or site B was deleted, the distribution of the mutants was diffuse (Fig. 3, B and C) , coinciding largely with that of CRT (Fig. 3, B′ and C′). Largely diffuse distribution was also obtained after site-specific mutation of full-length CSQ (Fig. 4 A) at three acidic amino acids in site B, D341A, E344A, and D345A (Fig. 4 C). In contrast, site-specific mutation of three, more proximal glutamic acid residues (E337A, E338A, and E340A) failed to affect the punctate distribution, which remained unchanged with respect to intact CSQ (Fig. 4 B).

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