Limits...
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.

Show MeSH

Related in: MedlinePlus

Notch signaling in Drosophila aph-1 mutants. (A and B) Neurogenic phenotype of embryos lacking maternal and zygotic aph-1 function (B) compared with wild type (A), using immunostaining for ELAV to visualize neuronal nuclei. Arrowhead in B indicates a small remaining patch of dorsal epidermis. (C–H) Clones of homozygous aph-1 mutant tissue exhibit Notch-like phenotypes, including notches and missing margin structures (arrowhead) in the wing (D), thoracic cuticle regions lacking microchaetae (F, arrowheads), and small eyes with disordered ommatidial packing (H). (I–N) SOP cell differentiation in larval imaginal wing discs from wild-type flies expressing no transgene (I), the membrane-tethered Notch fragment ΔECN (K), or the intracellular Notch fragment N(intra) (M), and from homozygous aph-1 mutant flies expressing no transgene (J), ΔECN (L), or N(intra) (N); aph-1 genotype indicated at bottom left, transgene listed at bottom right. Abbreviations: wp, wing pouch; marg, margin SOP cells; notum, region that develops into adult thorax; NO TG, no transgene.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199374&req=5

fig1: Notch signaling in Drosophila aph-1 mutants. (A and B) Neurogenic phenotype of embryos lacking maternal and zygotic aph-1 function (B) compared with wild type (A), using immunostaining for ELAV to visualize neuronal nuclei. Arrowhead in B indicates a small remaining patch of dorsal epidermis. (C–H) Clones of homozygous aph-1 mutant tissue exhibit Notch-like phenotypes, including notches and missing margin structures (arrowhead) in the wing (D), thoracic cuticle regions lacking microchaetae (F, arrowheads), and small eyes with disordered ommatidial packing (H). (I–N) SOP cell differentiation in larval imaginal wing discs from wild-type flies expressing no transgene (I), the membrane-tethered Notch fragment ΔECN (K), or the intracellular Notch fragment N(intra) (M), and from homozygous aph-1 mutant flies expressing no transgene (J), ΔECN (L), or N(intra) (N); aph-1 genotype indicated at bottom left, transgene listed at bottom right. Abbreviations: wp, wing pouch; marg, margin SOP cells; notum, region that develops into adult thorax; NO TG, no transgene.

Mentions: The postembryonic lethality of aph-1 mutants indicated that an embryonic requirement for Aph-1 in Notch signaling might be masked by maternally deposited protein. Therefore, we generated adult females bearing aph-1 mutant germline clones, and found that embryos lacking both maternal and zygotic Aph-1 activity exhibit a Notch-like hyperplasia of the embryonic nervous system (Fig. 1, A and B). Similarly, aph-1 mutant wing discs contain clusters of supernumerary sensory organ precursor (SOP) cells in addition to an overall developmental arrest phenotype, as seen in other neurogenic mutants (Fig. 1, I and J). To examine adult tissue phenotypes, we induced homozygous mutant aph-1 somatic tissue clones in heterozygous hosts. Numerous phenotypes characteristic of impaired Notch function were observed, including wing notching and bristle loss (Fig. 1, C–H). Together, these results demonstrate that Aph-1 is required for Notch signaling throughout development, and that aph-1 mutants show phenotypes indistinguishable from Psn and nct mutants.


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

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

Notch signaling in Drosophila aph-1 mutants. (A and B) Neurogenic phenotype of embryos lacking maternal and zygotic aph-1 function (B) compared with wild type (A), using immunostaining for ELAV to visualize neuronal nuclei. Arrowhead in B indicates a small remaining patch of dorsal epidermis. (C–H) Clones of homozygous aph-1 mutant tissue exhibit Notch-like phenotypes, including notches and missing margin structures (arrowhead) in the wing (D), thoracic cuticle regions lacking microchaetae (F, arrowheads), and small eyes with disordered ommatidial packing (H). (I–N) SOP cell differentiation in larval imaginal wing discs from wild-type flies expressing no transgene (I), the membrane-tethered Notch fragment ΔECN (K), or the intracellular Notch fragment N(intra) (M), and from homozygous aph-1 mutant flies expressing no transgene (J), ΔECN (L), or N(intra) (N); aph-1 genotype indicated at bottom left, transgene listed at bottom right. Abbreviations: wp, wing pouch; marg, margin SOP cells; notum, region that develops into adult thorax; NO TG, no transgene.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Notch signaling in Drosophila aph-1 mutants. (A and B) Neurogenic phenotype of embryos lacking maternal and zygotic aph-1 function (B) compared with wild type (A), using immunostaining for ELAV to visualize neuronal nuclei. Arrowhead in B indicates a small remaining patch of dorsal epidermis. (C–H) Clones of homozygous aph-1 mutant tissue exhibit Notch-like phenotypes, including notches and missing margin structures (arrowhead) in the wing (D), thoracic cuticle regions lacking microchaetae (F, arrowheads), and small eyes with disordered ommatidial packing (H). (I–N) SOP cell differentiation in larval imaginal wing discs from wild-type flies expressing no transgene (I), the membrane-tethered Notch fragment ΔECN (K), or the intracellular Notch fragment N(intra) (M), and from homozygous aph-1 mutant flies expressing no transgene (J), ΔECN (L), or N(intra) (N); aph-1 genotype indicated at bottom left, transgene listed at bottom right. Abbreviations: wp, wing pouch; marg, margin SOP cells; notum, region that develops into adult thorax; NO TG, no transgene.
Mentions: The postembryonic lethality of aph-1 mutants indicated that an embryonic requirement for Aph-1 in Notch signaling might be masked by maternally deposited protein. Therefore, we generated adult females bearing aph-1 mutant germline clones, and found that embryos lacking both maternal and zygotic Aph-1 activity exhibit a Notch-like hyperplasia of the embryonic nervous system (Fig. 1, A and B). Similarly, aph-1 mutant wing discs contain clusters of supernumerary sensory organ precursor (SOP) cells in addition to an overall developmental arrest phenotype, as seen in other neurogenic mutants (Fig. 1, I and J). To examine adult tissue phenotypes, we induced homozygous mutant aph-1 somatic tissue clones in heterozygous hosts. Numerous phenotypes characteristic of impaired Notch function were observed, including wing notching and bristle loss (Fig. 1, C–H). Together, these results demonstrate that Aph-1 is required for Notch signaling throughout development, and that aph-1 mutants show phenotypes indistinguishable from Psn and nct mutants.

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