Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism
- Kulandaivelu S Vetrivel†1,
- Ping Gong†1,
- James W Bowen†2,
- Haipeng Cheng1,
- Ying Chen1,
- Meghan Carter1,
- Phuong D Nguyen3,
- Lisa Placanica4,
- Felix T Wieland5,
- Yue-Ming Li4,
- Maria Z Kounnas3 and
- Gopal Thinakaran1, 2Email author
© Vetrivel et al; licensee BioMed Central Ltd. 2007
Received: 29 January 2007
Accepted: 08 February 2007
Published: 08 February 2007
Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides. Aβ is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by γ-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating γ-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated.
Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Aβ production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature γ-secretase complexes isolated using an active-site γ-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Aβ42 production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in γ-secretase modulation.
These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis.
Amyloid precursor protein (APP) is a type I membrane protein that is trafficked through the secretory and endocytic pathways in neuronal and non-neuronal cells, and is the precursor to 40–42 amino acid residue β-amyloid peptides (Aβ). Cerebral deposition of Aβ in senile plaques is a pathological feature of patients with Alzheimer's disease (AD), and Aβ deposits are also found in aged individuals. Aβ is liberated from APP via sequential proteolysis by β- and γ-secretases . Cleavage of APP within the lumenal domain by BACE1, the major neuronal β-secretase, releases the APP ectodomain and generates the N-terminus of Aβ . The APP ectodomain can also be released by cleavage at the "α-secretase" site within the Aβ domain by zinc metallopreotases such as TACE/ADAM17, ADAM9, ADAM10 and MDC-9, and an aspartyl protease BACE2 . The C-terminal APP stubs (APP CTFs) resulting from α – and β-secretase cleavage serve as substrates for intramembranous proteolysis by γ-secretase, a multimeric complex made of presenilin (PS) 1 or 2, nicastrin, APH1 and PEN2 . Mature components of the γ-secretase complex are found, and shown to be enzymatically active, at the cell surface as well as in multiple organelles such as the ER/Golgi intermediate compartment (ERGIC), Golgi apparatus, trans-Golgi network (TGN), and late endosomes . Activation of Notch signaling also involves sequential proteolytic processing, which closely resembles proteolysis of APP. Following ligand binding at the cell surface, Notch is endocytosed and sequentially cleaved by ADAM family metalloproteases and γ-secretase . Enhancer/suppressor screen studies in Caenorhabditis elegans originally suggested a role for p24 proteins in the transport regulation of Notch receptors to the cell surface . Reducing the activity of the p24 family member SEL-9 increased the cell surface accumulation of a transport-defective GLP-1 mutant, and increased the activity of mutant LIN-12 or GLP-1. Recently γ-secretase complex was found to contain p23 (also called TMP21), a p24 family protein. Intriguingly, reducing p23 expression resulted in increased γ-secretase cleavage of APP, without affecting the proteolysis of Notch .
The p24 proteins are a phylogenetically-conserved family of type I transmembrane proteins  that are highly enriched in the ER, Golgi, and coat protein (COP) I and II transport vesicles [10, 11]. Mammalian p24 family consists of six members, p23/TMP21, p24/p24a, p25/gp25L, p26/p24b, p27, and tp24, which function as hetero-oligomeric complexes . Sorting motifs in the cytosolic tail of p24 proteins bind to coat proteins of COPI and COPII vesicles [13–16], and ADP-ribosylation factor 1 (ARF1) . Furthermore, p23, p24a, and p25 are present in complexes with the Golgi reassembly stacking proteins GRASP55 and GRASP65 . A yeast strain lacking all eight members of the p24 family was viable displaying only minor secretory deficits , while in mice targeted disruption of both p23 alleles resulted in early embryonic lethality . Proposed roles of the p24 proteins include COP vesicle cargo receptors, regulators of COP vesicle budding, ER quality control, and organization of the Golgi apparatus [7, 13, 16, 21–25]. Recent evidence suggests that p24 proteins may specifically populate a subset of COPI vesicles  or influence the formation of tubular transport intermediates . Thus, their precise role in early secretory pathway trafficking, and more importantly sorting of specific cargo, still remains elusive.
Here, we investigated p23 modulation of APP metabolism and report that diminution of p23 expression in non-neuronal and neuroblastoma cell lines leads to increased biosynthetic stability and maturation of nascent APP, cell surface accumulation of APP, as well as secretion of sAPP. Selective increase in Aβ42 production associated with a familial AD (FAD)-linked PS mutation occurs independently of p23 modulation of APP metabolism. Finally, immunoisolation and immunofluorescence analysis reveals redistribution of APP and its adaptor Mint3/X11γ in cells with knockdown of p23 expression, suggesting a potential mechanism by which p23 may influence APP trafficking and sAPP release. Our data reveal a novel role for the p24 family proteins in regulating early secretory pathway trafficking of holo APP in addition to the recently reported modulation of γ-secretase cleavage of APP CTFs.
siRNA knockdown of p23 expression affects secretion of APPs and Aβ peptides in mouse neuroblastoma cells
Knockdown of p23 affects APP maturation and cell surface expression
Stable knockdown of p23 expression in HeLa cells increases endogenous sAPPα secretion
To address whether p23 knockdown influences cell-free generation of Aβ in HeLa cell membranes, we incubated C100FLAG substrate with membranes prepared from control and p23 knockdown clones using a well-established in vitro assay . To demonstrate the specificity in these assays, one set of each sample was incubated in parallel with the γ-secretase inhibitor L-685,458 to inhibit Aβ production. By ELISA analysis, we found that membrane preparations from p23 knockdown clones generated about 50% more Aβ40 relative to membranes from control clones (Fig. 4B). We then asked whether p23 is incorporated into active γ-secretase complexes. To this end, we used Merck C, a specific biotinylated transition-state γ-secretase inhibitor that is capable of capturing active γ-secretase complexes from membrane preparations . Interestingly, we found that the γ-secretase protein complexes captured from CHAPSO solubilized HeLa cell membranes contained readily detectable levels of PS1 NTF, but not p23 (Fig. 4E). As expected, we also detected PS1 CTF and nicastrin in γ-secretase complexes isolated using Merck C (data not shown). Moreover, addition of excess transition-state analog inhibitor L-685,458 prevented the capture, confirming selective isolation of active γ-secretase complexes by Merck C (Fig. 4E). These results indicate that p23 is not present at 1:1 stoichiometry with PS1 as a stable component of active γ-secretase complexes. Nevertheless, knockdown of p23 expression can influence in vitro Aβ production in a cell-free assay using an exogenous APP substrate and membrane preparations from HeLa (our results) or HEK293 cells , consistent with modulation of Aβ production by transient interaction of p23 with the mature γ-secretase complex.
Role of p23 in PS1 FAD mutation-associated selective increase of Aβ42secretion
Influence of p23 knockdown on the localization of APP and Mint3
Members of the p24 family proteins cycle between cis-Golgi and the ERGIC . Proposed functions of p24 proteins in ER/Golgi transport include integral receptors for COPI and COPII coatomer components, recruitment of ARF1 to Golgi membrane, Golgi organization, de novo formation of vesicular tubular clusters, generation of ER exit sites, and the formation of tubular transport intermediates [13, 14, 17, 20, 24, 27, 37]. Deletion of yeast p24 ortholog EMP24 impaired retention of Kar2p/BiP, indicative of defects in Golgi to ER retrieval . Moreover, partial defects in Golgi maturation of Gas1p (a glycosyl-phosphatidylinositol anchored plasma membrane protein) and secretion of invertase (a soluble protein that is secreted into the periplasmic space) in yeast p24 deletion mutants led to the suggestion that p24 proteins regulate cargo entry into COPII vesicles [19, 21]. Nevertheless, secretion of carboxypeptidase Y and pro α-factor were unaffected in yeast lacking all p24 proteins, arguing against an essential role in vesicular transport in the exocytic pathway . Consistent with this idea, our [35S]met/cys labeling studies reveal only minor decrease in overall protein biogenesis or secretion in N2a cells upon p23 knockdown.
Based on p23 antibody-mediated inhibition of vesicular stomatitis virus G protein trafficking, it was proposed that p23 plays an obligatory role in forward biosynthetic transport of transmembrane proteins in mammalian cells . In contrast, loss of SEL-9 (p24 ortholog) function in C. elegans increased cell surface trafficking of mutant but not wt GLP-1 (Notch homolog). Our data demonstrate enhanced APP maturation and cell surface accumulation in cells transfected with p23 siRNA. Moreover, we find an increase in the secretion of endogenous wt APP as well as stably overexpressed FAD-associated mutant APPswe following attenuation of p24 family protein expression. These findings are consistent with improved biosynthetic transport of APP to the cell surface in the absence of p23/p24 family function. In this regard, it is notable that PS1 deficiency also results in enhanced release of APP-containing vesicles from the ER and TGN, leading to increased maturation and cell surface accumulation of APP . Considering that packaging of APP and PS1 into COPII vesicles during ER export is uncoupled , it is highly likely that p23 depletion affects APP FL trafficking and PS1-dependent γ-secretase activity via distinct mechanisms. In agreement, we find that expression of FAD-linked mutant PS1 and depletion of p24 proteins have additive effects on Aβ42 production. One plausible mechanistic explanation for our results is that p23 function restricts biosynthetic trafficking of APP; p23-dependent APP retention/retrieval mechanisms operating in the continuum of bidirectional transport between the ER and Golgi are compromised upon p23 knockdown, allowing enhanced transit of nascent APP from the ER/Golgi through the exocytic pathway. Thus, our study provides the important insight that p23 functions in both positive and negative regulation of transmembrane cargo transport in the early secretory pathway. Elucidating the precise function of p23 in controlling the fidelity of APP trafficking remains a challenge because overexpression of p23 leads to retention of exogenous and endogenous p24 proteins in the ER, causing experimental artifacts (data not shown) [10, 24].
Several cytosolic adaptors with phosphotyrosine-binding domains, including Mint family proteins, Fe65 family members, and Dab1 bind to the APP cytoplasmic tail at or near the YENPTY motif, and regulate APP trafficking and processing . Mint proteins can directly bind to ARFs in a GTP-dependent manner, and siRNA knockdown of Mint3 resulted in diffuse localization of APP throughout the cell and concomitant decrease in juxtanuclear Golgi concentration . Thus it was postulated that Mints might serve as coat proteins in regulating vesicular trafficking of APP. We find that endogenous Mint3 localizes to the Golgi apparatus and shows colocalization with p23, β-COP and giantin. Depletion of p23 markedly reduced juxtanuclear concentration of Mint3 and membrane co-localization of APP and Mint3 evidenced by immunofluorescence and magnetic immunoisolation analyses (Fig. 6 and data not shown). Together, these results raise the intriguing possibility that Mint proteins may functionally link p23 to APP cargo and influence it's trafficking. Since Mints are recruited to the Golgi membrane by interaction with ARFs , and recruitment of ARF1 to the Golgi is mediated by p23 , it is reasonable to hypothesize that p23 influence on Mint3 localization may involve ARFs. This prediction awaits direct experimental confirmation. Molecular characterization of functional interactions between p23/ARF/Mint/APP is a focus of our future investigation. Finally, it is interesting to note that while the phosphotyrosine-binding domain of Mint proteins bind to APP cytosolic tail, the PDZ domains of Mint also bind to the C-terminus of PS1. Thus, functional interaction between p23/Mint/PS1 may be critically involved in the regulation of APP trafficking as well as γ-secretase processing.
Plasmids, oligonucleotides, and antibodies
Complementary oligonucleotides corresponding to three p23 target sequences separated by a 9-nucleotide non-complementary spacer (TTCAAGAGA) were synthesized and cloned into a modified pSUPER plasmid . One of the three siRNAs directed against a conserved sequence 5'-ATACCTGACCAACTCGTGA (nt 416–434 of NM_006827) present in human and mouse p23 mRNA, was identified as the most efficient by Western blotting and used for subsequent experiments. Synthetic siRNA duplexes against the above sequence were purchased from Dharmacon. A non-specific control siRNA was designed against the sequence CTGCAGAGCTCGACCACTC . The non-specific control shRNA plasmid was generated using oligonucleotides targeted to green fluorescence protein sequence.
Polyclonal p23 antisera were generated against a synthetic peptide ISFHLPVNSRKCLREEIHKDLLVTGA, corresponding to the N-terminal luminal residues 32–57 of mouse p23. Polyclonal Ab R8666 was raised against a synthetic peptide corresponding to the C-terminal 13 amino acids of APP and affinity purified. mAb B436 reacts with the amino-terminal region of Aβ and also recognizes sAPPα. mAb B113 and A387 were raised against Aβ sequences and are selective for Aβ40 and Aβ42, respectively. Antibodies against γ-secretase components , and p24 family proteins p24, p25, and tp24 have been described . APP C-terminal Ab 369 was provided by Drs. Sam Gandy (Farber Institute for Neurosciences, Philadelphia) and Huaxi Xu (The Burnham Institute, La Jolla). Polyclonal GRASP55 antiserum was a gift of Vivek Malhotra (University of California, San Diego). The following antibodies were purchased from commercial sources: mAb P2-1 against APP N-terminus (Biosource), mAb 5228 (Chemicon), calnexin (Stressgen), β-COP (Affinity Bioreagents, Inc.), giantin (Covance), p115 and Mint3 (BD Biosciences), OKT8 and 9E10 (ATCC).
Mouse N2a neuroblastoma cell lines expressing human APP695SWE and human PS1 have been previously described [29, 43]. HeLa/APPSWEDISH cells were provided by Gang Yu (UT Southwestern, Dallas) . HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin/streptomycin, and 2 mM L-glutamine (Invitrogen). N2a cells were maintained in the medium above mixed 1:1 with OptiMEM (Invitrogen). For transient p23 knockdown, cells were transfected twice with 200 or 300 nM p23 siRNA in 60 mm dishes and experiments were performed 72 h later. HeLa cells were stably transfected with pSUPERp23neo and the efficacy of p23 depletion in individual clones was determined by Western blot analysis.
Total cell lysates for immunoblotting were prepared in IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% sodium dexoycholate, 0.5% NP40, 0.25% SDS, 5 mM EDTA, 0.25 mM PMSF, and supplemented with a protease inhibitor cocktail [Sigma]). Metabolic labeling using [35S]met/cys and APP immunoprecipitation were performed as described . APP was immunoprecipitated from lysates and conditioned media using Ab 369 and mAb P2-1, respectively. Total protein synthesis and secretion were determined by using trichloroacetic acid precipitation combined with scintillation counting or by SDS/PAGE combined with phosphorimager detection. To measure cell surface APP, live cells were washed in cold HEPES buffer and blocked in HEPES buffer containing 0.1% BSA for 30 min at 4°C. Cells were then incubated at 10°C with mAb P2-1 for 90 min. After washing, cells were incubated for 3 h at 4°C with 1 μCi/ml 125I-conjuated anti-mouse secondary Ab (Amersham Biosciences). After extensive washes, cells were lysed in IP buffer and Ab binding was quantified by γ-counting. Non-specific binding was determined by omitting primary Ab, and specific binding was normalized to total protein concentration determined by BCA assay.
Aβ and sAPPα measurements
Fresh medium was added to cells 24 h after transfection with siRNA and conditioned media were collected at 48 h. The levels of secreted Aβ40, Aβ42, and sAPPα were quantified using specific sandwich ELISAs. Briefly, 96-well white ELISA plates were coated with the appropriate capture mAb (B113 for Aβ40, A387 for Aβ42, and 5228 for sAPPα). Following sample incubation for all three ELISAs, plates were washed and Aβ40, Aβ42 or sAPPα were detected with alkaline phosphatase-conjugated mAb B436 and CSPD-Sapphire II Luminescence Substrate (Applied Biosystems). Each sample was assayed in duplicate using appropriate dilution of the conditioned media so that the relative luminescent units were in the linear range of the standards included on each plate. For Aβ quantifications synthetic Aβ40 and Aβ42 peptides were diluted in culture medium to generate standard curve. sAPPα sample relative luminescence unit values were compared to a standard curve prepared from affinity-purified sAPPα. Briefly, conditioned medium from cells expressing human wild-type APP751 was collected and passed over an affinity column linked with a mAb that recognized sAPPα but not sAPPβ. Fractions were eluted with low pH and neutralized. A pooled sample containing the sAPPα peak was quantified by a protein assay and utilized for the sAPPα standard curve.
In vitroγ-secretase assays
Cell-free assays using C100FLAG substrate were performed essentially as described  using membranes prepared from non-specific control and p23 knockdown clones. Merck C, a highly specific biotinylated γ-secretase inhibitor , was used for affinity isolation of γ-secretase. The CHAPSO solubilized HeLa S3 membranes were incubated with 10 nM of Merck C for 2 h at 37°C in the absence or presence of 2 μM L-685,458. The samples were then incubated with streptavidin beads to capture inhibitor-bound γ-secretase complexes as previously described . Bound proteins were released by incubating in SDS-sample buffer and analyzed by immunoblotting.
Subcellular fractionation studies
Confluent cells from nine 60 mm dishes were homogenized using ball-bearing homogenizer with a 12 μm clearance and postnuclear supernatants were fractionated on sucrose density gradients essentially as described previously . Twelve 1 ml fractions were collected from the top of the gradient using a fractionator and 60 μl of each fraction was analyzed by Western blotting. Immunoisolation of APP containing vesicles using magnetic beads coated with mAb 9E10 was performed essentially as described . Antibody OKT8 was used as the negative control to establish the specificity of the immunoisolation procedure.
Cells cultured on poly-lysine coated coverslips were processed for immunofluorescence analysis as previously described . Primary antibodies were diluted in PBS containing 3% BSA and 0.2% Tween-20 and added to fixed cells at room temperature for 2 h. Images were acquired as 200 nm z-stacks on a motorized Nikon TE2000 microscope with Cascade II:512 CCD camera (Photometrics, Tucson, AZ) using 100× 1.45 NA Plan-Apochromat oil objective. Images were deconvolved using Huygens software (Scientific Volume Imaging BV, The Netherlands) and processed using Metamorph software (Molecular Devices Corporation, Downingtown, PA).
amyloid precursor protein
- APP FL:
ADP ribosylation factor 1
ER/Golgi intermediate compartment
short hairpin RNA
We thank Dr. Gang Yu for generously providing the HeLa/APPSWEDISH cell line; Drs. Sam Gandy, Huaxi Xu, and Vivek Malhotra for providing antibodies; Drs. Hiroshi Yajima and Kamal Sharma for providing pSUPER-GFP plasmid; and Dr. Daria Hazuda for Merck C γ-secretase inhibitor. This work is supported by NIH grants to GT (AG021495 and AG019070) and Y-ML (AG026660), and grants from the Alzheimer's Association (IIRG to GT; NIRG to KSV; Zenith Fellows Award to Y-ML), and American Health Assistance Foundation (GT and Y-ML).
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