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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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Summary chart and hypothetical γ-secretase assembly pathway. (A) Chart summarizing the design and major outcomes of the coexpression experiments. Epitope tags on Nct, Aph-1, and Pen-2 are indicated by underlining. (B) Hypothetical model of γ-secretase assembly and activation. Nct and Aph-1 associate in an early subcomplex, leading to stabilization of Aph-1. The subcomplex regulates stabilization of Psn holoprotein and its incorporation into a complex with Nct and possibly Pen-2. Pen-2 is required for endoproteolysis of Psn holoprotein, although the point at which Pen-2 enters the pathway is unknown.
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fig5: Summary chart and hypothetical γ-secretase assembly pathway. (A) Chart summarizing the design and major outcomes of the coexpression experiments. Epitope tags on Nct, Aph-1, and Pen-2 are indicated by underlining. (B) Hypothetical model of γ-secretase assembly and activation. Nct and Aph-1 associate in an early subcomplex, leading to stabilization of Aph-1. The subcomplex regulates stabilization of Psn holoprotein and its incorporation into a complex with Nct and possibly Pen-2. Pen-2 is required for endoproteolysis of Psn holoprotein, although the point at which Pen-2 enters the pathway is unknown.

Mentions: Because Drosophila S2 cells faithfully replicate the stringently regulated, low level accumulation of mature Psn seen in mammalian cells (Hu et al., 2002), we used them to test whether coexpression of all four putative γ-secretase components promotes the assembly of mature γ-secretase. However, we found that certain combinations of epitope-tagged components were unstable, particularly those involving coexpression of tagged Nct and tagged Aph-1 (see Fig. 5 A). Tagged Pen-2 was relatively well tolerated in most cases (see Fig. 5 A). Therefore, we initially expressed Psn-myc with untagged Nct, Aph-1, and/or Pen-2, monitoring only the levels of Psn holoprotein and COOH-terminal fragment in each combination. To monitor the levels of the other components, these works were repeated using Psn-myc with just one other epitope-tagged component (e.g., Aph-1-V5, Nct-myc, or Pen-2-flag). Levels of Psn-myc were monitored in these samples to verify that each component combination gave reproducible effects on Psn, whether other components were epitope-tagged or not. Using this strategy, we obtained consistent results for Psn levels without apparent complications due to epitope tags.


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

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

Summary chart and hypothetical γ-secretase assembly pathway. (A) Chart summarizing the design and major outcomes of the coexpression experiments. Epitope tags on Nct, Aph-1, and Pen-2 are indicated by underlining. (B) Hypothetical model of γ-secretase assembly and activation. Nct and Aph-1 associate in an early subcomplex, leading to stabilization of Aph-1. The subcomplex regulates stabilization of Psn holoprotein and its incorporation into a complex with Nct and possibly Pen-2. Pen-2 is required for endoproteolysis of Psn holoprotein, although the point at which Pen-2 enters the pathway is unknown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199374&req=5

fig5: Summary chart and hypothetical γ-secretase assembly pathway. (A) Chart summarizing the design and major outcomes of the coexpression experiments. Epitope tags on Nct, Aph-1, and Pen-2 are indicated by underlining. (B) Hypothetical model of γ-secretase assembly and activation. Nct and Aph-1 associate in an early subcomplex, leading to stabilization of Aph-1. The subcomplex regulates stabilization of Psn holoprotein and its incorporation into a complex with Nct and possibly Pen-2. Pen-2 is required for endoproteolysis of Psn holoprotein, although the point at which Pen-2 enters the pathway is unknown.
Mentions: Because Drosophila S2 cells faithfully replicate the stringently regulated, low level accumulation of mature Psn seen in mammalian cells (Hu et al., 2002), we used them to test whether coexpression of all four putative γ-secretase components promotes the assembly of mature γ-secretase. However, we found that certain combinations of epitope-tagged components were unstable, particularly those involving coexpression of tagged Nct and tagged Aph-1 (see Fig. 5 A). Tagged Pen-2 was relatively well tolerated in most cases (see Fig. 5 A). Therefore, we initially expressed Psn-myc with untagged Nct, Aph-1, and/or Pen-2, monitoring only the levels of Psn holoprotein and COOH-terminal fragment in each combination. To monitor the levels of the other components, these works were repeated using Psn-myc with just one other epitope-tagged component (e.g., Aph-1-V5, Nct-myc, or Pen-2-flag). Levels of Psn-myc were monitored in these samples to verify that each component combination gave reproducible effects on Psn, whether other components were epitope-tagged or not. Using this strategy, we obtained consistent results for Psn levels without apparent complications due to epitope tags.

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
Related in: MedlinePlus