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Targeting of inositol 1,4,5-trisphosphate receptor to the endoplasmic reticulum by its first transmembrane domain.

Pantazaka E, Taylor CW - Biochem. J. (2009)

Bottom Line: Replacement of these post-TMD1 residues with unrelated sequences of similar length (24-36 residues) partially mimicked the native residues.We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER.Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK.

ABSTRACT
Targeting of IP3R (inositol 1,4,5-trisphosphate receptors) to membranes of the ER (endoplasmic reticulum) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of IP3R1 (type 1 IP3R) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2 (second transmembrane domain). Replacement of these post-TMD1 residues with unrelated sequences of similar length (24-36 residues) partially mimicked the native residues. We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER. Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

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Fusion proteins used(A) Key regions of IP3R1. (B) Sequence of the TMD1-2 region. Numbers in parentheses denote the number of residues within each region. (C) The proteins used, and their abbreviations, are shown with N-terminal EGFP (black) or EYFP (grey) tags represented as ovals, and the C-terminal FLAG epitope as a flag. TMD are shown by black bars, linking loops by white bars, and the N- and C-termini by hatching.
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Figure 1: Fusion proteins used(A) Key regions of IP3R1. (B) Sequence of the TMD1-2 region. Numbers in parentheses denote the number of residues within each region. (C) The proteins used, and their abbreviations, are shown with N-terminal EGFP (black) or EYFP (grey) tags represented as ovals, and the C-terminal FLAG epitope as a flag. TMD are shown by black bars, linking loops by white bars, and the N- and C-termini by hatching.

Mentions: IP3R [IP3 (inositol 1,4,5-trisphosphate) receptors] are the intracellular Ca2+ channels that both initiate and regeneratively propagate the cytosolic Ca2+ signals evoked by the many receptors that stimulate IP3 formation [1]. All IP3R are tetramers, each with an IP3-binding site lying close to the N-terminus and six TMD (transmembrane domains) lying close to the C-terminus (Figure 1A). The last pair of TMD from each subunit together with their intervening luminal loop form the pore [2,3]. In most animal cells, IP3R are expressed mainly within the membranes of the ER (endoplasmic reticulum), but they are also expressed within the nuclear envelope [4], nucleoplasmic reticulum [5], Golgi apparatus [6], plasma membrane [7], and perhaps also in secretory vesicles [8], although the latter is contentious [9]. Within these membranes, IP3R are not uniformly distributed and different subtypes may differ in their distributions [10,11]. The subcellular distribution of IP3R accounts for their ability to generate cytosolic Ca2+ signals that are spatially organized, thereby allowing Ca2+ to regulate specifically a diverse array of cellular processes [1]. The versatility of Ca2+ as a ubiquitous intracellular messenger thus depends upon precise targeting of IP3R to specific subcellular compartments.


Targeting of inositol 1,4,5-trisphosphate receptor to the endoplasmic reticulum by its first transmembrane domain.

Pantazaka E, Taylor CW - Biochem. J. (2009)

Fusion proteins used(A) Key regions of IP3R1. (B) Sequence of the TMD1-2 region. Numbers in parentheses denote the number of residues within each region. (C) The proteins used, and their abbreviations, are shown with N-terminal EGFP (black) or EYFP (grey) tags represented as ovals, and the C-terminal FLAG epitope as a flag. TMD are shown by black bars, linking loops by white bars, and the N- and C-termini by hatching.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805921&req=5

Figure 1: Fusion proteins used(A) Key regions of IP3R1. (B) Sequence of the TMD1-2 region. Numbers in parentheses denote the number of residues within each region. (C) The proteins used, and their abbreviations, are shown with N-terminal EGFP (black) or EYFP (grey) tags represented as ovals, and the C-terminal FLAG epitope as a flag. TMD are shown by black bars, linking loops by white bars, and the N- and C-termini by hatching.
Mentions: IP3R [IP3 (inositol 1,4,5-trisphosphate) receptors] are the intracellular Ca2+ channels that both initiate and regeneratively propagate the cytosolic Ca2+ signals evoked by the many receptors that stimulate IP3 formation [1]. All IP3R are tetramers, each with an IP3-binding site lying close to the N-terminus and six TMD (transmembrane domains) lying close to the C-terminus (Figure 1A). The last pair of TMD from each subunit together with their intervening luminal loop form the pore [2,3]. In most animal cells, IP3R are expressed mainly within the membranes of the ER (endoplasmic reticulum), but they are also expressed within the nuclear envelope [4], nucleoplasmic reticulum [5], Golgi apparatus [6], plasma membrane [7], and perhaps also in secretory vesicles [8], although the latter is contentious [9]. Within these membranes, IP3R are not uniformly distributed and different subtypes may differ in their distributions [10,11]. The subcellular distribution of IP3R accounts for their ability to generate cytosolic Ca2+ signals that are spatially organized, thereby allowing Ca2+ to regulate specifically a diverse array of cellular processes [1]. The versatility of Ca2+ as a ubiquitous intracellular messenger thus depends upon precise targeting of IP3R to specific subcellular compartments.

Bottom Line: Replacement of these post-TMD1 residues with unrelated sequences of similar length (24-36 residues) partially mimicked the native residues.We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER.Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK.

ABSTRACT
Targeting of IP3R (inositol 1,4,5-trisphosphate receptors) to membranes of the ER (endoplasmic reticulum) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of IP3R1 (type 1 IP3R) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2 (second transmembrane domain). Replacement of these post-TMD1 residues with unrelated sequences of similar length (24-36 residues) partially mimicked the native residues. We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER. Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

Show MeSH
Related in: MedlinePlus