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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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Activity of overproduced γ-secretase assessed by Notch processing. Coexpression of all four putative γ-secretase components is required for enhanced production of the Psn-dependent p120 fragment of the Notch receptor. Two replicated S2 cell transfection experiments are shown (total, n = 14), in which combinations of three different components or all four were coexpressed with intact Notch. Lysates were resolved by SDS-PAGE and immunoprobed using antibodies that detect the cleaved COOH-terminal fragments of Notch in the 120-kD range. For samples involving only three components, similar amounts of the three Notch COOH-terminal fragments are detected (bands 1–3, left), as in cells expressing 0–2 components (not depicted). Coexpression of all four components leads to significant overproduction of the second 120-kD species (γ, right), corresponding to the γ-secretase–dependent 120-kD cleaved fragment of Notch.
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fig4: Activity of overproduced γ-secretase assessed by Notch processing. Coexpression of all four putative γ-secretase components is required for enhanced production of the Psn-dependent p120 fragment of the Notch receptor. Two replicated S2 cell transfection experiments are shown (total, n = 14), in which combinations of three different components or all four were coexpressed with intact Notch. Lysates were resolved by SDS-PAGE and immunoprobed using antibodies that detect the cleaved COOH-terminal fragments of Notch in the 120-kD range. For samples involving only three components, similar amounts of the three Notch COOH-terminal fragments are detected (bands 1–3, left), as in cells expressing 0–2 components (not depicted). Coexpression of all four components leads to significant overproduction of the second 120-kD species (γ, right), corresponding to the γ-secretase–dependent 120-kD cleaved fragment of Notch.

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)

Activity of overproduced γ-secretase assessed by Notch processing. Coexpression of all four putative γ-secretase components is required for enhanced production of the Psn-dependent p120 fragment of the Notch receptor. Two replicated S2 cell transfection experiments are shown (total, n = 14), in which combinations of three different components or all four were coexpressed with intact Notch. Lysates were resolved by SDS-PAGE and immunoprobed using antibodies that detect the cleaved COOH-terminal fragments of Notch in the 120-kD range. For samples involving only three components, similar amounts of the three Notch COOH-terminal fragments are detected (bands 1–3, left), as in cells expressing 0–2 components (not depicted). Coexpression of all four components leads to significant overproduction of the second 120-kD species (γ, right), corresponding to the γ-secretase–dependent 120-kD cleaved fragment of Notch.
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Related In: Results  -  Collection

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fig4: Activity of overproduced γ-secretase assessed by Notch processing. Coexpression of all four putative γ-secretase components is required for enhanced production of the Psn-dependent p120 fragment of the Notch receptor. Two replicated S2 cell transfection experiments are shown (total, n = 14), in which combinations of three different components or all four were coexpressed with intact Notch. Lysates were resolved by SDS-PAGE and immunoprobed using antibodies that detect the cleaved COOH-terminal fragments of Notch in the 120-kD range. For samples involving only three components, similar amounts of the three Notch COOH-terminal fragments are detected (bands 1–3, left), as in cells expressing 0–2 components (not depicted). Coexpression of all four components leads to significant overproduction of the second 120-kD species (γ, right), corresponding to the γ-secretase–dependent 120-kD cleaved fragment of Notch.
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