Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex

Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis.     

Topology of the C-terminal fragment of human presenilin 1

Mutations of human presenilin 1 (PS1) have been genetically linked to early-onset familial Alzheimer's disease. PS1 contains 10 hydrophobic regions (HRs) sufficiently long to be alpha-helical membrane spanning segments. Most previous topology studies agree that the N-terminus of PS1 is cytosolic and HRs 1-6 span the membrane but HR 7 does not. However, whether HRs 8 and 9 are membrane spanning segments remains controversial. Here we study the topology and biogenesis of this region of PS1 using a reporter gene fusion approach, where portions of the PS1 sequence containing possible membrane spanning segments were fused up- or downstream of a reporter sequence whose translocation into the endoplasmic reticulum could be monitored via its glycosylation. We provide strong evidence, supported by cysteine accessibility studies in full-length PS1, that HRs 8 and 9 are indeed membrane spanning and that the integration of HR 8 into the membrane is dependent on the presence of HR 9. We also explain how our results reconcile previous apparently divergent conclusions regarding the topology of HRs 8 and 9.     

Membrane topology and nicastrin-enhanced endoproteolysis of APH-1, a component of the gamma-secretase complex

APH-1, presenilin, nicastrin, and Pen-2 are proteins with varying membrane topologies that compose the gamma-secretase complex, which is responsible for the intramembrane proteolysis of several substrates including the amyloid precursor protein. APH-1 is known to be necessary for gamma-secretase activity, but its precise function in the complex is not fully understood, and its membrane topology has not been described, although it is predicted to traverse the membrane seven times. To investigate this, we used selective permeabilization of the plasma membrane and immunofluorescence microscopy to show that the C terminus of the APH-1 resides in the cytosolic space. Insertion of N-linked glycosylation sites into each of the hydrophilic loop domains and the N terminus of APH-1 showed that the N-terminal domain as well as loops 2, 4, and 6 could be glycosylated, whereas loops 1, 3, and 5 were not. Thus, APH-1 topologically resembles a seven-transmembrane domain receptor with the N terminus and even-numbered loops facing the endoplasmic reticulum lumen, and the C terminus and odd-numbered loops reside in the cytosolic space. By using these glycosylation mutants, we provide evidence that the association between nicastrin and APH-1 may occur very soon after APH-1 synthesis and that the interaction between these two proteins may rely more heavily on the transmembrane domains of APH-1 than on the loop domains. Furthermore, we found that APH-1 can be processed by several endoproteolytic events. One of these cleavages is strongly up-regulated by co-expression of nicastrin and generates a stable C-terminal fragment that associates with nicastrin.     

The transmembrane domain region of nicastrin mediates direct interactions with APH-1 and the gamma-secretase complex

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.     

A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.     

Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway

The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.     

The extreme C terminus of presenilin 1 is essential for gamma-secretase complex assembly and activity

The gamma-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid beta-peptide and the cytoplasmic APP intracellular domain. The active gamma-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for gamma-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, gamma-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease gamma-secretase activity, PS1 endoproteolysis, and gamma-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.     

Presenilin 1 stabilizes the C-terminal fragment of the amyloid precursor protein independently of gamma-secretase activity

The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1.     

Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.     

Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1

Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.     

Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.     

The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.

The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.     

Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55 degrees C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The beta subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the alpha subunit were extruded, while the rest of alpha retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of alpha was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of alpha subunits as dimers, trimers, and tetramers without beta or gamma subunits. This implies specific alpha-alpha contact. Third, the gamma subunit, like the C-terminal spans of alpha, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of alpha, beta, and gamma subunits in which alpha can associate in assemblies as large as tetramers via its cytoplasmic domain, while beta and gamma subunits associate with alpha primarily in its C-terminal portion, which has a unique structure and thermal instability.     

Contribution of presenilin transmembrane domains 6 and 7 to a water-containing cavity in the gamma-secretase complex

Gamma-secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of gamma-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective gamma-secretase inhibitors could be designed.     

Pen-2 is incorporated into the gamma-secretase complex through binding to transmembrane domain 4 of presenilin 1

gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. We reconstituted PS1 in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). We report that the proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, are required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, we further demonstrate that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1.     

Calsenilin is a substrate for caspase-3 that preferentially interacts with the familial Alzheimer's disease-associated C-terminal fragment of presenilin 2

Calsenilin is a member of the recoverin family of neuronal calcium-binding proteins that we have previously shown to interact with presenilin 1 (PS1) and presenilin 2 (PS2) holoproteins. The expression of calsenilin can regulate the levels of a proteolytic product of PS2 (Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) Nat. Med. 4, 1177-1181) and reverse the presenilin-mediated enhancement of calcium signaling (Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8590-8593). Here, we have used cultured mammalian cells that transiently or stably express calsenilin to extend the characterization of calsenilin and of the calsenilin-PS2 interaction. We have found that calsenilin has the ability to interact with endogenous 25-kDa C-terminal fragment (CTF) that is a product of regulated endoproteolytic cleavage of PS2 and that the presence of the N141I PS2 mutation does not significantly alter the interaction of calsenilin with PS2. Interestingly, when the 25-kDa PS2 CTF and the 20-kDa PS2 CTF are both present, calsenilin preferentially interacts with the 20-kDa CTF. Increases in the 20-kDa fragment are associated with the presence of familial Alzheimer's disease-associated mutations (Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997) Science 277, 373-376). However, the finding that the production of the 20-kDa fragment is regulated by the phosphorylation of PS2 (Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1391-1396) suggests that it is a regulated physiological event that also occurs in the absence of the familial Alzheimer's disease-associated mutations in PS2. Finally, we have demonstrated that calsenilin is a substrate for caspase-3, and we have used site-directed mutagenesis to map the caspase-3 cleavage site to a region that is proximal to the calcium binding domain of calsenilin.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.     

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.     

Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-beta precursor protein and Notch

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.     

The subunit structure of the follitropin receptor. Chemical cross-linking of the solubilized follitropin-receptor complex

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.