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PubMed2003

Cardiac L-type calcium channel beta-subunits expressed in human heart have differential effects on single channel characteristics.

l-Type calcium channels are multiprotein complexes composed of pore-forming (CaV1.2) and modulatory auxiliary alpha2delta- and beta-subunits. We demonstrate expression of two different isoforms for the beta2-subunit (beta2a, beta2b) and the beta3-subunit (beta3a, beta3trunc) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of beta2b transcripts prevails in the order of > beta3 >> beta2a. The expressed cardiac full-length beta3-subunit is identical to the beta3a-isoform, and beta3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium beta3trunc expression increases whereas overall beta3 expression remains unchanged. Heterologous coexpression studies demonstrated that beta2 induced larger currents through rabbit and human cardiac CaV1.2 pore subunits than beta3 isoforms. All beta-subunits increased channel availability at single channel level, but beta2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac beta-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various beta-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure.

Animals↗

PubMed2003

The Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42.

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The voltage-dependent calcium channel beta subunit contains two stable interacting domains.

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.

Amino Acid Motifs↗

PubMed2003

Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'-ends.

Messenger RNA 3'-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.

Carrier Proteins↗

PubMed2003

Identification of a novel TATA element-binding protein binding region at the N terminus of the Saccharomyces cerevisiae TAF1 protein.

TFIID, a multiprotein complex composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), can directly recognize core promoter elements and mediate transcriptional activation. The TAF N-terminal domain (TAND) of TAF1 may play a significant role in these two principal TFIID functions by regulating the access of TBP to the TATA element. In yeast, TAND consists of two subdomains, TAND1 (10-37 amino acids (aa)) and TAND2 (46-71 aa), which interact with the concave and convex surfaces of TBP, respectively. Here we demonstrate that another region located on the C-terminal side of TAND2 (82-139 aa) can also bind to TBP and induce transcriptional activation when tethered to DNA as a GAL4 fusion protein. As these properties are the same as those of TAND1, we denoted this sequence as TAND3. Detailed mutational analyses revealed that three blocks of hydrophobic amino acid residues located within TAND3 are required not only for TBP binding and transcriptional activation but also for supporting cell growth and the efficient transcription of a subset of genes. We also show that the surface of TBP recognized by TAND3 is broader than that recognized by TAND1, although these regions overlap partially. Supporting these observations is that TAND1 can be at least partly functionally substituted by TAND3.

Alanine↗

PubMed2003

Polycomb group suppressor of zeste 12 links heterochromatin protein 1alpha and enhancer of zeste 2.

Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3. Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 showed not only homeotic transformation but also position effect variegation. We now report that the mammalian SU(Z)12 directly interacts with heterochromatin protein 1alpha (HP1alpha) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo. Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1alpha (amino acid residues 479-536) and the VEFS box for EZH2 (amino acid residues 600-639), which are not mutually exclusive. Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly. In addition SU(Z)12 represses transcription activity in the presence of HP1alpha in a reporter assay. These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12.

Amino Acid Sequence↗

PubMed2003

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53.

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.

Animals↗

PubMed2003

The Kinesin-related protein Costal2 associates with membranes in a Hedgehog-sensitive, Smoothened-independent manner.

In Drosophila, Hedgehog (Hh) signal transduction has been shown to require a multiprotein complex (Hedgehog signaling complex (HSC)), which includes the Kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). We present evidence that a biologically relevant fraction of the HSC is found in association with cellular membranes. We demonstrate that Cos2 is capable of tethering an exogenous protein to vesicular membranes and that Cos2 association with membranes is Hh-sensitive. In addition, we demonstrate that Cos2 associates with membranes in cells that lack the transmembrane protein Smoothened (Smo) through a domain of Cos2 distinct from its recently characterized Smo binding domain. We suggest that an Hh-regulated membrane binding activity of Cos2 is part of the mechanism by which Cos2 contributes to Hh signaling. We propose a model in which there are two distinct HSCs with discrete subcellular localizations and activities: one is endosome-associated and facilitates production of a repressor form of Ci (HSC-R), and one is Smo-associated and promotes Ci activation (HSC-A). In response to Hh and through interaction with Cos2, Smo mediates both inhibition of the endosome-associated HSC-R and activation of HSC-A at the plasma membrane.

Animals↗

PubMed2004

Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium.

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Animals↗

PubMed2004

MINT, the Msx2 interacting nuclear matrix target, enhances Runx2-dependent activation of the osteocalcin fibroblast growth factor response element.

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/Cbfa1. To better understand Msx2 regulation of the OCFRE, we have studied functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naïve CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses.

Amino Acid Motifs↗

PubMed2004

Tight junction protein Par6 interacts with an evolutionarily conserved region in the amino terminus of PALS1/stardust.

Tight junctions are the structures in mammalian epithelial cells that separate the apical and basolateral membranes and may also be important in the establishment of cell polarity. Two evolutionarily conserved multiprotein complexes, Crumbs-PALS1 (Stardust)-PATJ and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. These two complexes have been linked physically and functionally by an interaction between PALS1 and Par6. Here we identify an evolutionarily conserved region in the amino terminus of PALS1 as the Par6 binding site and identify valine and aspartic acid residues in this region as essential for interacting with the PDZ domain of Par6. We have also characterized, in more detail, the amino terminus of Drosophila Stardust and demonstrate that the interaction mechanism between Stardust and Drosophila Par6 is evolutionarily conserved. Par6 interferes with PATJ in binding PALS1, and these two interactions do not appear to function synergistically. Taken together, these results define the molecular mechanisms linking two conserved polarity complexes.

Amino Acid Sequence↗

PubMed2004

Clathrin-mediated endocytosis of m3 muscarinic receptors. Roles for Gbetagamma and tubulin.

Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether Galpha(q) or Gbetagamma undergo such regulated translocation. Recent studies demonstrate that m3 muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates Galpha(q) and activates phospholipase Cbeta1. Interaction of tubulin-GDP with Gbetagamma at the offset of phospholipase Cbeta1 signaling appears involved in translocation of tubulin and Gbetagamma to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299-34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m3 muscarinic receptors or Galpha(q) involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of Galpha(q), Gbetagamma, tubulin, clathrin, and m3 receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, Galpha(q) and Gbetagamma. Fifteen minutes later receptors, Gbetagamma and tubulin, but not Galpha(q), internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m3 receptors with Gbetagamma, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that Gbetagamma subunits might organize the formation of a multiprotein complex linking m3 receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but beta-arrestin-independent endocytosis of m3 muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other beta-arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.

Blotting, Western↗

PubMed2004

A tissue-specific, naturally occurring human SNF2L variant inactivates chromatin remodeling.

Mammalian genomes encode two imitation switch family chromatin remodeling proteins, SNF2H and SNF2L. In the mouse, SNF2H is expressed ubiquitously, whereas SNF2L expression is limited to the brain and gonadal tissue. This pattern of SNF2L expression suggests a critical role for SNF2L in neuronal physiology. Indeed, SNF2L was shown to promote neurite outgrowth as well as regulate the human engrailed homeotic genes, important regulators of brain development. Here we identify a novel splice variant of human SNF2L we call SNF2L+13, which contains a nonconserved in-frame exon within the conserved catalytic core domain of SNF2L. SNF2L+13 retains the ability to incorporate into multiprotein complexes; however, it is devoid of enzymatic activity. Most interestingly, unlike mouse SNF2L, human SNF2L is expressed ubiquitously, and regulation is mediated by isoform variation. The human SNF2L+13 null variant is predominant in non-neuronal tissue, whereas the human wild type active SNF2L isoform is expressed in neurons. Thus, like the mouse, active human SNF2L is limited to neurons and a few other tissues.

Adenosine Triphosphatases↗

PubMed2004

Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling.

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic check-point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.

Animals↗

PubMed2004

Identification of a novel partner of duox: EFP1, a thioredoxin-related protein.

H(2)O(2) is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid. ThOX proteins (thyroid oxidase) also called Duox are believed to be responsible for H(2)O(2) generation. Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions. In this study, we demonstrate interactions of Duoxs with TPO and with p22(phox) without any effect on Duox activity. By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1 (EF-hand binding protein 1), one partner of Duoxs that belongs to the thioredoxin-related protein family. EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved. EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes. It interacts also with TPO but not thyroglobulin. Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H(2)O(2) production. EFP1 and Duox mRNA share similar distribution in nine different tissues. These results suggest that EFP1 could be one of the partners in the assembly of the multiprotein complex constituting the thyroid H(2)O(2) generating system but is certainly not sufficient to permit H(2)O(2) generation.

Amino Acid Sequence↗

PubMed2004

Structural insights into the mechanism of formation of cellulosomes probed by small angle X-ray scattering.

Exploring the mechanism by which the multiprotein complexes of cellulolytic organisms, the cellulosomes, attain their exceptional synergy is a challenge for biologists. We have studied the solution structures of the Clostridium cellulolyticum cellulosomal enzyme Cel48F in the free and complexed states with cohesins from Clostridium thermocellum and Clostridium cellulolyticum by small angle x-ray scattering in order to investigate the conformational events likely to occur upon complexation. The solution structure of the free cellulase indicates that the dockerin module is folded, whereas the linker connecting the catalytic module to the dockerin is extended and flexible. Remarkably, the docking of the different cohesins onto Cel48F leads to a pleating of the linker. The global structure determined here allowed modeling of the atomic structure of the C. cellulolyticum dockerin-cohesin interface, highlighting the local differences between both organisms responsible for the species specificity.

Amino Acid Sequence↗

PubMed2004

Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis.

The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable cross-linker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immunoblotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGh/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.

Bacterial Proteins↗

PubMed2005

Nicastrin is critical for stability and trafficking but not association of other presenilin/gamma-secretase components.

gamma-Secretase, which is responsible for the intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/gamma-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/gamma-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and full-length PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the gamma-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved full-length PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2.PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/ gamma-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1.

Amyloid Precursor Protein Secretases↗