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Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex.

Hu Y, Fortini ME - J. Cell Biol. (2003)

Bottom Line: Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex.In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly.These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.

View Article: PubMed Central - PubMed

Affiliation: University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

ABSTRACT
The gamma-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative gamma-secretase components have been identified-presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in gamma-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in gamma-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.

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Effects of combinatorial expression of putative γ-secretase components on Psn, Nct, Aph-1, and Pen-2 levels. (A) Different combinations of putative γ-secretase components were expressed in S2 cells, as indicated above each blot (+, present; −, absent), and cells were harvested and examined for levels of the immature Psn holoprotein (Psn holo) and mature Psn COOH-terminal fragment (Psn CTF), using a myc-tagged form of Psn. Samples were examined for endogenous β-tubulin and a cotransfected GFP plasmid as loading/transfection controls. In this experiment, all other transfected components (Nct, Aph-1, and/or Pen-2) were not epitope-tagged. Asterisks (rightmost lanes) denote pairs of components that were expressed together from two different cotransfected plasmids. (B) To monitor levels of each of the other coexpressed γ-secretase components, the experiment in A was repeated, using Psn-myc together with one other tagged component and two untagged components for each replicate (left, Nct-myc with untagged Aph-1 and Pen-2; middle, Pen-2-flag with untagged Nct and Aph-1; right, Aph-1-V5 with untagged Nct and Pen-2; asterisks as in A). For clarity, uninformative 1- and 2-component lanes of the Nct-myc and Pen-2-flag blots are omitted. Informative 1- and 2-component samples are included for the Aph-1-V5 immunoblot; the two-plasmid expression lanes of the Aph-1-V5 blot are omitted because this approach always produces a low level of residual Aph-1-V5 that is not seen when all four components are expressed from a single plasmid, and which we ascribe to a small percentage of cells that are not transfected by both plasmids. (C) Coexpression performed with a nonfunctional form of Nct with an internal deletion of residues 304–333.
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fig3: Effects of combinatorial expression of putative γ-secretase components on Psn, Nct, Aph-1, and Pen-2 levels. (A) Different combinations of putative γ-secretase components were expressed in S2 cells, as indicated above each blot (+, present; −, absent), and cells were harvested and examined for levels of the immature Psn holoprotein (Psn holo) and mature Psn COOH-terminal fragment (Psn CTF), using a myc-tagged form of Psn. Samples were examined for endogenous β-tubulin and a cotransfected GFP plasmid as loading/transfection controls. In this experiment, all other transfected components (Nct, Aph-1, and/or Pen-2) were not epitope-tagged. Asterisks (rightmost lanes) denote pairs of components that were expressed together from two different cotransfected plasmids. (B) To monitor levels of each of the other coexpressed γ-secretase components, the experiment in A was repeated, using Psn-myc together with one other tagged component and two untagged components for each replicate (left, Nct-myc with untagged Aph-1 and Pen-2; middle, Pen-2-flag with untagged Nct and Aph-1; right, Aph-1-V5 with untagged Nct and Pen-2; asterisks as in A). For clarity, uninformative 1- and 2-component lanes of the Nct-myc and Pen-2-flag blots are omitted. Informative 1- and 2-component samples are included for the Aph-1-V5 immunoblot; the two-plasmid expression lanes of the Aph-1-V5 blot are omitted because this approach always produces a low level of residual Aph-1-V5 that is not seen when all four components are expressed from a single plasmid, and which we ascribe to a small percentage of cells that are not transfected by both plasmids. (C) Coexpression performed with a nonfunctional form of Nct with an internal deletion of residues 304–333.

Mentions: S2 cells were transfected with constructs encoding stepwise combination of the four γ-secretase components. No massive overproduction of Psn holoprotein or COOH-terminal fragments was observed in samples involving up to three coexpressed proteins. However, coexpression of all four proteins together results in high levels of mature γ-secretase, as indicated by dramatically elevated levels of cleaved Psn COOH-terminal fragments and tagged Pen-2 (Fig. 3, A and B). These effects were paralleled by a significant increase in the specific Notch p120 cleavage attributed to γ-secretase, implying an elevated production of functional γ-secretase (Fig. 4). Nct levels remain relatively constant in all samples, including those involving coexpression of all four proteins (Fig. 3 B). However, Aph-1 accumulates to high levels only when coexpressed with Nct or Nct with Pen-2, as described later (Fig. 3 B).


Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex.

Hu Y, Fortini ME - J. Cell Biol. (2003)

Effects of combinatorial expression of putative γ-secretase components on Psn, Nct, Aph-1, and Pen-2 levels. (A) Different combinations of putative γ-secretase components were expressed in S2 cells, as indicated above each blot (+, present; −, absent), and cells were harvested and examined for levels of the immature Psn holoprotein (Psn holo) and mature Psn COOH-terminal fragment (Psn CTF), using a myc-tagged form of Psn. Samples were examined for endogenous β-tubulin and a cotransfected GFP plasmid as loading/transfection controls. In this experiment, all other transfected components (Nct, Aph-1, and/or Pen-2) were not epitope-tagged. Asterisks (rightmost lanes) denote pairs of components that were expressed together from two different cotransfected plasmids. (B) To monitor levels of each of the other coexpressed γ-secretase components, the experiment in A was repeated, using Psn-myc together with one other tagged component and two untagged components for each replicate (left, Nct-myc with untagged Aph-1 and Pen-2; middle, Pen-2-flag with untagged Nct and Aph-1; right, Aph-1-V5 with untagged Nct and Pen-2; asterisks as in A). For clarity, uninformative 1- and 2-component lanes of the Nct-myc and Pen-2-flag blots are omitted. Informative 1- and 2-component samples are included for the Aph-1-V5 immunoblot; the two-plasmid expression lanes of the Aph-1-V5 blot are omitted because this approach always produces a low level of residual Aph-1-V5 that is not seen when all four components are expressed from a single plasmid, and which we ascribe to a small percentage of cells that are not transfected by both plasmids. (C) Coexpression performed with a nonfunctional form of Nct with an internal deletion of residues 304–333.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199374&req=5

fig3: Effects of combinatorial expression of putative γ-secretase components on Psn, Nct, Aph-1, and Pen-2 levels. (A) Different combinations of putative γ-secretase components were expressed in S2 cells, as indicated above each blot (+, present; −, absent), and cells were harvested and examined for levels of the immature Psn holoprotein (Psn holo) and mature Psn COOH-terminal fragment (Psn CTF), using a myc-tagged form of Psn. Samples were examined for endogenous β-tubulin and a cotransfected GFP plasmid as loading/transfection controls. In this experiment, all other transfected components (Nct, Aph-1, and/or Pen-2) were not epitope-tagged. Asterisks (rightmost lanes) denote pairs of components that were expressed together from two different cotransfected plasmids. (B) To monitor levels of each of the other coexpressed γ-secretase components, the experiment in A was repeated, using Psn-myc together with one other tagged component and two untagged components for each replicate (left, Nct-myc with untagged Aph-1 and Pen-2; middle, Pen-2-flag with untagged Nct and Aph-1; right, Aph-1-V5 with untagged Nct and Pen-2; asterisks as in A). For clarity, uninformative 1- and 2-component lanes of the Nct-myc and Pen-2-flag blots are omitted. Informative 1- and 2-component samples are included for the Aph-1-V5 immunoblot; the two-plasmid expression lanes of the Aph-1-V5 blot are omitted because this approach always produces a low level of residual Aph-1-V5 that is not seen when all four components are expressed from a single plasmid, and which we ascribe to a small percentage of cells that are not transfected by both plasmids. (C) Coexpression performed with a nonfunctional form of Nct with an internal deletion of residues 304–333.
Mentions: S2 cells were transfected with constructs encoding stepwise combination of the four γ-secretase components. No massive overproduction of Psn holoprotein or COOH-terminal fragments was observed in samples involving up to three coexpressed proteins. However, coexpression of all four proteins together results in high levels of mature γ-secretase, as indicated by dramatically elevated levels of cleaved Psn COOH-terminal fragments and tagged Pen-2 (Fig. 3, A and B). These effects were paralleled by a significant increase in the specific Notch p120 cleavage attributed to γ-secretase, implying an elevated production of functional γ-secretase (Fig. 4). Nct levels remain relatively constant in all samples, including those involving coexpression of all four proteins (Fig. 3 B). However, Aph-1 accumulates to high levels only when coexpressed with Nct or Nct with Pen-2, as described later (Fig. 3 B).

Bottom Line: Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex.In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly.These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.

View Article: PubMed Central - PubMed

Affiliation: University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

ABSTRACT
The gamma-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative gamma-secretase components have been identified-presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in gamma-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in gamma-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.

Show MeSH