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Originally published In Press as doi:10.1074/jbc.M411091200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7758-7768, March 4, 2005
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Uncoupled Packaging of Amyloid Precursor Protein and Presenilin 1 into Coat Protein Complex II Vesicles*

Jinoh Kim{ddagger}, Susan Hamamoto{ddagger}, Mariella Ravazzola§, Lelio Orci§, and Randy Schekman{ddagger}

From the {ddagger}Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720 and §Department of Cellular Physiology and Metabolism, University of Geneva Medical School, 1211 Geneva 4, Switzerland

Received for publication, September 27, 2004 , and in revised form, December 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Presenilin (PS)1 is a polytopic membrane protein mutant alleles of which are linked to early onset Alzheimer's disease (AD) (AD mutation data base,). Several lines of evidence are consistent with the idea that PS serves as a trafficking regulator of other membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. PS1 is localized in the ER and in early Golgi membranes in the steady state where it may guide the transport of TrkB and make physical contact with Notch and amyloid precursor protein (APP) (1–5). By analogy in the cholesterol regulatory pathway, SREBP (sterol regulatory element-binding protein) traffic and proteolytic activation are guided by SCAP (SREBP cleavage-activating protein) (for review, see Ref. 6). Similarly, PS may somehow guide the traffic and proteolytic cleavage of APP by the {alpha}-, {beta}-, and {gamma}-secretases. A biochemical analysis of the traffic of PS and APP from the ER would permit an evaluation of their transport interdependence or independence.

Sec23/24p, Sec13/31p, and Sar1p are the cytosolic coat proteins, called COPII, responsible for the formation of ER-to-Golgi anterograde transport vesicles (7). The action of COPII proteins is confined to the ER membrane due to a specific interaction of the GTP-binding protein, Sar1p, with the ER-bound GTP exchange factor for Sar1p, Sec12p (8, 9). The GTP-bound form of Sar1p sequentially recruits the Sec23/24p complex and the Sec13/31p complex to the ER membrane, resulting in deformation of the membrane and packaging of cargo proteins into transport vesicles. Sec23p functions as a GTPase-activating protein for Sar1p (10). Sec13/31p further stimulates the GTPase-activating protein activity of Sec23p about 10-fold, resulting in the release of coat proteins from the membranes (11). Thus, maintenance of the coat on the membrane is affected by Sec12p, Sec23/24p, and Sec13/31p (12). Sec24p possesses multiple cargo binding sites to package diverse membrane proteins into COPII vesicles (13, 14). Of the COPII proteins, Sec24p has the largest number of homologs in organisms ranging from yeast to mammals, likely serving to expand the repertoire of cargo molecules captured for transport (13, 15). Sec16p, a peripheral membranes protein, interacts with Sec23p, Sec24p, and Sec31p (16, 17). Sec16p appears to stabilize a coat assembly intermediate (18).

COPII vesicle formation has been reconstituted using enriched mammalian proteins (19, 20). Basic principles of COPII vesicle formation processes are well conserved in the mammalian system (19, 20). To probe the regulatory role of PS in APP trafficking from ER to Golgi, we established a vesicle budding reaction and compared the activity of crude cytosol with purified recombinant mammalian COPII proteins expressed in a baculovirus system. We present evidence that a precursor form of PS1 and APP is independently packaged into COPII vesicles.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—PS1+/– fibroblast cells, PS1–/– fibroblast cells, PS+/+ embryonic stem cells, and PS–/– embryonic stem cells were gifts from S. Sisodia (University of Chicago, Chicago, IL). The pET11dHis-Sar1a vector was a gift of R. Pepperkok (EMBL, Heidelberg, Germany). Plasmids pFLAG-mSec23A and pHis-hSec24C were gifts of P. Espenshade (University of Texas Southwestern Medical Center, Dallas, TX). Plasmid GSTrbetI{Delta}TM was a gift of J. Hay (University of Michigan, Ann Arbor, MI). Plasmid pSG5-APP was a gift of W. Annaert (Catholic University of Leuven, Leuven, Belgium). The plasmid, pETGEXCT, encoding GST was described elsewhere (21). Plasmids pBSK-hSec13 and KIAA0905 were gifts of W. Hong (Institute of Molecular and Cell Biology, Singapore). Sequencing of the recombinant Sec24C gene (KIAA0079, accession number 1723050) revealed a guanosine base missing at codon 1041, which resulted in a frameshift from codon 1041. However, the resultant amino acid sequence was more homologous to the mouse Sec24C than the original sequence and was identical to the amino acid sequence deduced from the recently sequenced full-length human Sec24C cDNA sequence (accession number AAH18928 [GenBank] .

Rabbit muscle creatine phosphokinase, creatine phosphate, thrombin, ATP, and GTP and protease inhibitor mixture tablets were purchased from Roche Applied Science). Rabbit anti-ribophorin I serum was a kind gift from T. Rapoport (Harvard Medical School, Boston, MA); rabbit anti-p58 was from J. Saraste (University of Bergen, Bergen, Norway); rabbit anti-Sec23A antibody and anti-Sec24C antibody were gifts from J.-P. Paccaud (Geneva University, Geneva, Switzerland); rabbit anti-Sec13 antibody and anti-Sec31A antibody were gifts from W. Hong (Institute of Molecular and Cell Biology, Singapore); rabbit anti-APP C-terminal IgG fraction was purchased from Sigma (catalog number A8717); rabbit anti-PS1 antibody was a gift from S. Sisodia (University of Chicago, Chicago, IL); protein A-horseradish peroxidase was from Sigma (catalog number P8651); S35-labeled anti-rabbit IgG was from AP Biotech (catalog number SJ434, Piscataway, NJ); peroxidase-conjugated anti-His6 mouse monoclonal antibody was from Roche Applied Science (catalog number 1965085, Mannheim, Germany).

Most phospholipids and derivatives were purchased from Avanti Polar Lipid (Alabaster, AL). Inositol 4-phosphate, inositol diphosphate, CDP-diacylglycerol, and ergosterol were obtained from Sigma. SYPRO Red protein stain dye and Texas Red phosphatidylethanolamine were purchased from Molecular Probes.

Plasmid Constructions—The gene encoding hamster Sar1a was PCR-amplified from the pET11dHis-Sar1a vector using synthetic oligonucleotides JK1 (5'-CGTGGATCCATGTCCTTCATATTTG-3') and JK2 (5'-CAGTACATCGATTAGGAATTCATC-3'). The amplified fragments were digested with BamHI and EcoRI and inserted into corresponding sites of pTY40 (22), resulting in pJK1 (GST-hamster Sar1a fusion construct).

The human Sec13-coding region was amplified from pBSK-hSec13 using synthetic oligonucleotides JK28 (5'-ATCGGATCCATGTACCCATACGATGTTCCAGATTACGCTATGGTGTCAGTAATTAAC-3') and JK29 (5'-TAATACGACTCACTATAGG-3'). The amplified fragments were digested with BamHI and HindIII and inserted into the corresponding sites of pFLAG-mSec23A to replace Sec23 with Sec13. The resultant plasmid, pJK4, encodes hemagglutinin-tagged human Sec13.

The human Sec31A-coding region was amplified from KIAA0905 using synthetic oligonucleotides JK30 (5'-ATAGGATCCATGAAGTTAAAGGAAGTAG-3') and JK31 (5'-CCTCACTAAAGGGAAC-3'). The amplified fragments were digested with BamHI and NotI and inserted into the corresponding sites of pHis-hSec24C to replace Sec24C with Sec31A. This plasmid, pJK5 has a His6 tag at the N terminus.

The APP C-tail-coding region was amplified from pSG5-APP using synthetic oligonucleotides JK90 (5'-ATCACCGGATCCATGCTGAAGAAGAAACAGTAC-3') and JK91 (5'-CTCCTCGAATTCCTAGTTCTGCATCTGCTCAAA-3'). The amplified fragments were digested with BamHI and EcoRI and inserted into corresponding sites of pTY40, resulting in pJK11 (GST-APPct fusion construct).

Synthetic oligonucleotides JK92 (5'-TACATCGGATCCAGGACTCAGCAAGAAGCAGCTGCCAAAAAATTCTTCTGAGAATTCGAGGAG-3') and JK93 (5'-CTCCTCGAATTCTCAGAAGAATTTTTTGGCAGCTGCTTCTTGCTGAGTCCTGGATCCGATGTA-3') were annealed and digested with BamHI and EcoRI and inserted into the corresponding sites of pTY40, yielding a GST-p58ct fusion construct (pJK12).

Buffers—Phosphate-buffered saline without calcium and magnesium (PBS, pH 7.4) was purchased from Mediatech (catalog number 21–031-CV, Herndon, VA). Buffer C consists of 10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10% (w/v) SDS plus protease inhibitor mixture. Buffer D consists of 150 mM Tris-HCl (pH 6.8), 15% SDS, 25% (v/v) glycerol, 0.02% (w/v) bromphenol blue, and 12.5% (v/v) 2-mercaptoethanol. Buffer E consists of 50 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 70 mM potassium acetate, 5 mM potassium EGTA, 0.5 mM magnesium acetate plus protease inhibitor mixture. Buffer F consists of 10 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 10 mM potassium acetate, 1.5 mM magnesium acetate plus protease inhibitor mixture. Buffer G consists of 20 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 150 mM potassium acetate, 0.5 mM magnesium acetate plus protease inhibitor mixture. Buffer H contains 25 mM HEPES (pH 7.2), 150 mM potassium acetate, 10% glycerol, 250 mM sorbitol, 1 mM EDTA, and 1 mM EGTA. TBS contains 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 10 µM GDP. TBST is TBS supplemented with 0.1% Tween 20. TCB contains 50 mM Tris-HCl (pH 8.0), 250 mM potassium acetate, 5 mM CaCl2, and 10 µM GDP. HKM contains 20 mM HEPES-KOH (pH 6.8), 160 mM potassium acetate, and 5 mM MgCl2. Buffer BB contains 20 mM HEPES (pH 7.2), 300 mM potassium acetate, 5% glycerol, 5 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol.

Hamster Sar1a Purification—Overnight cultures of Escherichia coli BL21(DE3) carrying pJK1 or pJK2 were diluted into 1 liter of 2x YT (16 g bacto tryptone, 10 g bacto yeast extract, and 5 g NaCl per liter) medium (1/1000). The cells were grown at 37 °C to an A600 of 0.3, transferred to 25 °C, and further grown to an A600 of 0.8. Expression of hamster Sar1a was induced with 0.1 mM isopropyl 1-thio-{beta}-D-galactopyranoside for 2 h. The induced cells were harvested by centrifugation at 4500 rpm for 25 min in a Sorvall RC3B rotor at 4 °C. The pellet was resuspended with 25 ml of TBST and treated with lysozyme (0.8 mg/ml) for 20 min at RT with occasional mixing. After incubation Triton X-100 was added to a final concentration of 1%. The cells were sonicated with three pulses of 30 s. Cell debris was removed by successive centrifugations at 1500 rpm in a Sorval SS34 rotor for 30 min and at 40,000 rpm in a Beckman 70Ti rotor for 1 h. The supernatant was mixed with TBST-washed glutathione-Sepharose 4B beads (AP Biotech) and incubated for 1 h at 4 °C. After incubation the beads were washed three times with ice-cold TBST, once with TBS, and once with TCB. The beads were transferred into a disposable column and incubated with 4 units of thrombin (Roche Applied Science) for 90 min. Eluates containing hamster Sar1a cleaved from GST fusion protein were collected and dialyzed with HKM supplemented with 5 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µM GDP, and 5% glycerol. Proteins were quantified by the Bradford assay using bovine serum albumin as a standard (23).

Mammalian Sec23A/24C and Sec13/31A Purification—Recombinant insect baculovirus stocks were generated according to the manufacturer's protocol using Sf9 insect cells (Invitrogen). Recombinant mouse Sec23A/human 24C and human Sec13/31A complexes were purified essentially as described by Espenshade et al. (24). Sec23A copurifies with Sec24C, and Sec13 copurifies with Sec31A.

Yeast COPII Purification—Yeast Sar1p, Sec23/24p, and Sec13/31p were prepared as described previously (22, 25).

Purification of GST Fusion Proteins—GST or GST fusion proteins were purified as described for hamster Sar1a purification, except that bound GST fusion proteins were eluted with HKM containing 10 mM reduced glutathione. The eluates were dialyzed against HKM supplemented with 5 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 5% glycerol.

Gel Exclusion Chromatography—Aliquots (100 µl) of purified protein samples were applied to a Superose 6 column (10 x 300 mm, AP Biotech) at a flow rate of 0.1 ml/min in a buffer containing 20 mM HEPES (pH 8.0), 500 mM potassium acetate, 10% glycerol, 250 mM sorbitol, 5 mM 2-mercaptoethanol, and 0.1 mM EGTA at 4 °C. The column was calibrated with a mixture of globular molecular weight markers. Each of the purified COPII proteins filter as a single monodisperse species. Kav = (Ve – Vo)/(Vt x Vo), where Ve is an elution volume, Vo is a void volume, and Vt is a total volume.

Cell Culture—Chinese hamster ovary (CHO-K1) cells were maintained in monolayer at 37 °C in an atmosphere of 5% CO2 in medium A (a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 µg/ml streptomycin sulfate) supplemented with 5% (v/v) fetal bovine serum. Mouse fibroblast cells and embryonic stem cells were maintained as described by Sato et al. (26).

Preparation of Rat Liver Cytosol—Rat liver cytosol was prepared in Buffer E as described previously (27, 28).

Cytosol Fractionation with Ammonium Sulfate—Appropriate amounts of ammonium sulfate were serially added to rat liver cytosol to achieve 30, 40, 50, 60, 70, and 80% saturation. At each step precipitated proteins were harvested by centrifugation at 15,000 rpm for 30 min in a Sorval SS34 rotor at 4 °C. The volume of supernatant was measured, and additional ammonium sulfate was added to achieve the next level of saturation. Pellets at each ammonium sulfate concentration were solubilized with a small volume of buffer H and supplemented with 1 µg/ml calpain inhibitor, 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and 5 mM {beta}-mercaptoethanol. Insoluble aggregates were removed by centrifugation at 15,000 rpm for 30 min in a Sorval SS34 rotor at 4 °C. The cleared supernatants were dialyzed against buffer H.

Isolation of Microsomes—Cells grown to 70–80% confluency on 10-cm dishes were washed once with PBS, scraped into PBS (5 ml/dish), pooled, and harvested at 1000 x g for 5 min at 4 °C. The pellet was resuspended in PBS (0.5 ml/dish) and centrifuged again at 1000 x g for 5 min at 4 °C. The pellet was then resuspended in Buffer F (0.2 ml/dish), passed through a 22-gauge needle 20 times, and centrifuged at 1000 x g for 5 min at 4 °C. The post nuclear supernatant fraction was removed into siliconized microcentrifuge tubes, and microsomes were sedimented at 6000 x g for 10 min at 4 °C. Harvested microsomes were washed twice with Buffer G (0.1 ml/dish), and the pellet was resuspended in Buffer G (30 µl/dish). Thirty microliters of microsomes was used for each in vitro vesicle-formation reaction (see "In Vitro Transport Vesicle-formation Assay").

Liposome Binding Assay—Liposomes were prepared from the majorminor mix lipid formulation as described by Matsuoka and Schekman (29). For a mammalian COPII binding experiment, 15 µg of a liposome suspension was mixed with various combinations of hamster Sar1a (1.2 µg), Sec23A/24C (1.7 µg), and Sec13/31A (2.4 µg) in 75 µl of HKM buffer with or without 0.1 mM GMP-PNP. For a yeast COPII binding experiment, 1.1 µg of Sar1p, 1.5 µg of Sec23/24p, and 2.2 µg of Sec13/31p were used. After incubation for 25 min at 27 °C, the reaction mixture was diluted with 50 µl of HKM containing 2.5 M sucrose. An aliquot (110 µl) of this mixture was loaded into a polycarbonate centrifuge tube (Beckman catalog number 343775) and overlaid with 100 µl of HKM containing 0.75 M sucrose and 20 µl of HKM. The resulting step gradient was centrifuged at 100,000 rpm in a Beckman TLA-100 rotor for 20 min at RT. A sample (25 µl) was collected from the top of the tube. Proteins in the fraction were resolved by SDS-PAGE, stained by SYPRO Red, and visualized using a Typhoon 9400 image analyzer.

Liposome Budding Assay—Detailed procedures for the liposome budding assay were described in Matsuoka and Schekman (29). The liposomes contained 58 mol % dioleoylphosphatidylcholine, 22 mol % dioleoylphosphatidylethanolamine, 3 mol % dioleoylphosphatidylserine, 1 mol % dioleoyl phosphatidic acid, 10 mol % PI, 2 mol % CDP-diacylglycerol, 3 mol % sphingomyelin, 8 mol % cholesterol. Budding reactions contained 8.4 µg of Sar1a, 9.5 µg of Sec23A/24C, and 10.3 µg of Sec13/31A in 100 µl in the presence of GDP or GMP-PNP. The COPII vesicle fractions were separated using a sucrose-density gradient.

Microsome Binding Assay—About 25 µg of microsomes were mixed with various combinations of 0.4 µg of Sar1a, 0.42 µg of Sec23A/24C, 0.6 µg of Sec13/31A, 0.1 mM GMP-PNP, and 1x ATP regenerating system. After incubation at RT for 15 min, unless otherwise indicated 45 µl of the reaction mixture was layered on 150 µl of buffer G containing 0.3 M sucrose and centrifuged at 80,000 rpm in a Beckman TLA-100 rotor for 10 min at RT. The pellet was washed with 200 µl of buffer G, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Strips of membrane were probed with peroxidase-conjugated anti-His6 antibody and visualized by ECL plus. Where indicated microsomes were incubated with 1 M potassium acetate or 2.5 M urea in buffer G for 10 min and further washed in buffer G before the binding assay.

In Vitro Transport Vesicle-formation Assay—Each reaction contained 20 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 150 mM potassium acetate, 0.5 mM magnesium acetate, protease inhibitor mixture (1x), 25–50 µg of microsomes in a 100-µl final volume. Where indicated an ATP regenerating system (40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 1 mM ATP), 0.2 mM GTP, and cytosol or purified recombinant COPII proteins (1 µg of Sar1a, 1 µg of Sec23A/Sec24C, and 1 µg of Sec13/31A) were added. Reactions were preincubated on ice for 5 min, initiated at 25 °C, and terminated by transferring tubes to ice after 60 min. A 75-µl aliquot of the vesicle fraction (S14) was separated from the donor microsomal fraction (P14) by centrifugation at 14,000 x g for 20 min at 4 °C. The vesicles from the S14 were collected by centrifugation at 50,000 rpm at 4 °C in a Beckman TLA100 rotor for 20 min (P100). P14 and P100 fractions were solubilized with 60 and 16 µl of Buffer C, respectively, and supplemented with 15 and 4 µl of Buffer D, respectively. The resulting samples were heated at 42 °C for 15 min. Aliquots of P14 (15 µl; 20% of total) and P100 (20 µl; 75% of total) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and analyzed by immunoblotting.

GST Pull-down Assay—Sec23A/24C or Sec23A (5 µg) were incubated with 30 µg of GST fusion proteins in 100 µl of buffer BB containing 15 µl of prewashed GSHA beads (AP Biotech) and 10% lipid emulsion for 90 min at RT (30). Beads were washed three times with buffer BB containing 0.05% octyl glucoside, once with 20 mM Hepes (pH 7.2), and then eluted in SDS sample buffer. Eluates were analyzed by SDS-PAGE.

Immunoblotting—Antibodies were diluted as follows: anti-ribophorin I serum, 1/3,000; anti-p58 serum, 1/1,000; anti-APP C-terminal IgG, 1/2,000; anti-PS1 antibody, 1/20,000; anti-Sec23A antibody, 1/1,000; anti-Sec24C antibody, 1/1,000; anti-Sec13 antibody, 1/1,000; anti-Sec31A antibody, 1/1,000; protein A-horseradish peroxidase (1 mg/ml in PBS supplemented with 50% glycerol), 1/2,500; S35-labeled anti-rabbit IgG, 1/1,000. Membranes bound with primary antibodies were incubated with protein A-horseradish peroxidase for visualization using ECL plus (AP Biotech) or S35-labeled anti-rabbit secondary antibodies for quantitation using phosphorimaging (Typhoon 9400, AP Biotech). Peroxidase-conjugated anti-His6 antibody was diluted 1/500.

Electron Microscopy—In vitro vesicle formation assays were performed as described above. Membranes were sedimented by centrifugation at 55,000 rpm in a Beckman TLA-100.3 rotor for 25 min, fixed with 1% glutaraldehyde in buffer G for 30 min at 4 °C, and processed as described previously for conventional electron microscopy of thin sections (31).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purified mammalian Sec23A/24C complex (Fig. 1A) migrated on a Superose 6 gel with an apparent molecular mass of about 50 kDa (Fig. 1B). Considering the molecular masses of the recombinant Sec23A and Sec24C deduced from the cDNAs (88.3 and 122.5 kDa, respectively), Sec23A/24C appeared to interact with the matrix, resulting in longer retention during the chromatography. Mammalian Sec13/31A (36.8 and 136.4 kDa, respectively) eluted with an apparent molecular mass of 500 kDa, and yeast Sec13/31p eluted as a 700-kDa species (Fig. 1B). The elongated structure of tetrameric yeast Sec13/31p likely caused anomalous behavior on gel filtration chromatography (32). This behavior was also observed for the mammalian Sec13/31A, which eluted earlier than predicted for a heterotetramer (346.4 kDa).



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  		FIG. 1.
Interactions among purified recombinant COPII proteins on synthetic liposomes. A, purified protein complexes were separated by SDS-PAGE. B, oligomeric status of purified protein complexes was probed using Superose 6 gel permeation chromatography. Mammalian protein complexes are indicated with an m, whereas yeast counterparts are indicated with a y. Peak positions were marked with arrows. C, liposomes (major-minor mix) were incubated with COPII proteins plus nucleotides. Liposome-bound proteins were separated from unbound fractions using a sucrose step gradient and were resolved by SDS-PAGE. Asterisks represent degraded proteins.

 
To test whether the mammalian proteins preserved the signature interactions among COPII proteins that had been established with the yeast proteins, we performed a liposome flotation assay. GMP-PNP, a non-hydrolyzable GTP analogue, increased hamster Sar1a binding to the synthetic liposomes relative to the GDP state (Fig. 1C, lanes 1, 2, 8, and 9). The small increment may be due to the fact that hamster Sar1a has a slower GTP exchange rate than yeast Sar1p on major-minor liposomes (data not shown). Recruitment of mammalian Sec13/31A required Sar1a, GMP-PNP, and Sec23A/24C, consistent with the behavior of the yeast proteins. This strongly suggests that the recombinant mammalian proteins are functionally active and preserve interfaces for proper interactions between them (Fig. 1C) (33).

Next, we monitored the recruitment of the recombinant COPII proteins to microsomal membranes (Fig. 2). We took advantage of the fact that the recombinant Sec24C and Sec31A could be specifically recognized by an anti-His6 antibody. Sec24C was more efficiently recognized by the anti-His6 antibody than Sec31A for unknown reasons (Fig. 2A, lane 12). Sec24C was recruited to microsomes in a Sar1a and GMP-PNP-dependent manner. GTP was unable to maintain coat proteins on the membranes most likely because Sec23A, like its yeast counterpart, acts as a GTPase-activating protein for Sar1a (20). Upon GTP hydrolysis, coat proteins are released from the synthetic liposome (11). Recruitment of Sec31A was slightly enhanced by prolonged incubation times (compare lanes 7 and 15 of Fig. 2B).



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  		FIG. 2.
Recombinant mammalian COPII proteins are recruited to microsomal membranes. A and B, COPII proteins bound to CHO-K1 derived microsomes were sedimented from unbound proteins through a 0.3 M sucrose cushion and resolved by SDS-PAGE, then transferred to a polyvinylidene difluoride membrane. The His-tagged recombinant Sec24C and Sec31A were detected using a peroxidase-conjugated anti-His6 mouse monoclonal antibody. Note that there was a small amount of nonspecific binding of COPII proteins to the reaction tubes (see lane 11 in A). A, incubations were performed for 15 min at RT. C, microsomes were washed with high salt or urea. Binding assays were performed as in B. KOAc, potassium acetate; r, (an ATP) regenerating system.

 
An ATP regenerating system stimulated binding of the recombinant proteins to microsomal membranes (Fig. 2B). The stimulation of COPII binding to the membranes by ATP has been previously demonstrated, yet the molecule(s) responsible for the stimulation of COPII binding has not been found (34). Because this ATP requirement was observed with high salt- or urea-stripped membranes, the ATP utilizing factor may be an integral membrane protein (Fig. 2C, lane 3, 4, 11, and 12). The recombinant COPII proteins also showed an ATP requirement for efficient binding comparable with the endogenous counterparts. Thus, this aspect of the functional integrity of the recombinant purified proteins was not altered. In prolonged incubations, cytosol enhanced recruitment of purified proteins to microsomal membranes (Fig. 2B, lanes 13–20). This cytosol effect was more obvious when the membranes were stripped with high salt or urea (Fig. 2C). Unwashed membranes may contain a small amount of the cytosolic factor(s) that enhances COPII binding. Cytosol may simply stabilize binding of COPII to the membranes. The enhancement of COPII binding to membranes by cytosol did not require ATP (Fig. 2C compare lane 3 and 7, 11 and 15). We conclude that COPII binding can be independently modulated by ATP and cytosol.

An essential function of COPII proteins is to deform membranes into transport vesicles. We incubated synthetic liposomes (extruded with a 400 nm filter, Fig. 3B) with mammalian COPII proteins, and the reaction mixture was sedimented on a linear sucrose-density gradient to separate coated vesicles from uncoated liposomes. The sedimentation of lipids was monitored by measuring the fluorescence of Texas Red phosphatidylethanolamine incorporated into the major-minor mixture. The recovery of the lipids in the high density peak (fractions 11–13) was increased with addition of GMP-PNP in the reaction mixture (Fig. 3A). Thin section images of fractions 11–13 revealed coated vesicles that were similar to those of yeast COPII vesicles (Fig. 3, C and D). Mammalian COPII proteins generated larger vesicles (mean diameter without the coat 84.4 ± 16.1 nm; n = 100; p < 0.0001) than yeast COPII proteins (68.3 ± 17.4 nm; n = 100). The mammalian COPII proteins are, thus, able to deform large liposomes into small vesicles.



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  		FIG. 3.
Recombinant COPII proteins generate coated vesicles from synthetic liposomes. A, liposome budding reactions were sedimented on a linear sucrose gradient. Thin section electron microscopy images of starting liposomes (B) or COPII vesicle fractions (11 to 13) (C, mammalian COPII; D, yeast COPII). The coat is more apparent at higher magnification in the insets.

 
Cytosol and COPII proteins were incubated with microsomes. We performed a large scale (500 µl) incubation and measured transport vesicle formation by differential centrifugation. COPII vesicles were found in the S14 fraction separated from bulk membrane. Vesicles (S14) were collected by ultracentrifugation (P100) and processed for electron microscopy. The diameters of vesicles (without the coat) generated with cytosol were slightly larger (58.7 ± 10.9 nm; n = 200; p < 0.0005) (Fig. 4A) than those generated with purified COPII proteins (55.6 ± 10.3 nm; n = 200) (Fig. 4B). The purified mammalian COPII proteins yielded fewer vesicles than cytosol (23 ± 1 vesicles/µm2 S.E. versus 78 ± 4 vesicles/µm2; n = 30 pictures at magnification x 130548; p < 0.0001), although the reaction with purified proteins contained more COPII than the cytosol (Fig. 4, compare A and B). However, we observed many COPII cage-like structures in the thin section images generated with recombinant COPII proteins (Fig. 4B) (35). A fraction of vesicles generated by cytosol may be contributed by COPI vesicles derived by budding from Golgi membranes present in the microsomal membrane fraction.



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  		FIG. 4.
Vesicles generated with cytosol or COPII. Transport vesicles were generated using GTP and an ATP regenerating system in a 500-µl reaction. Cytosol (A) or purified recombinant mammalian COPII proteins (B) were used as a source of coat proteins. The vesicles are largely uncoated. In B "cage-like" coat structures independent of the vesicles are apparent. Vesicles were concentrated by centrifugation from an S14 fraction. These vesicles were processed for thin section electron microscopy, and the diameters of the vesicles were measured.

 
We then monitored packaging of cargo molecules into COPII vesicles using the transport vesicle formation assay. Cargo proteins in the vesicle fractions (P100) were identified by immunoblotting of p58, the equivalent of ERGIC53 (LMAN1), and APP. p58 is a particularly useful cargo molecule because it cycles between the ER and Golgi with a steady state localization in the ER and the intermediate compartment (36, 37). In contrast, APP transits to the cell surface, and in the steady state only a fraction resides in the ER (1). Cytosol stimulated the packaging of p58 and APP with only negligible capture of ribophorin I, a resident ER membrane protein (Fig. 5A, lanes 6–8). Packaging of p58 and APP was inhibited by a GDP-restricted form of Sar1a T39N, suggesting COPII vesicles are responsible for cargo packaging (see Fig. 7A). These results are consistent with previous reports on mammalian ER transport reactions (24, 27). However, purified COPII proteins showed very low cargo packaging activity compared with cytosol alone (Fig. 5A, compare lane 4 with lanes 6 and 7). Because cytosol enhanced binding of recombinant COPII proteins to microsomal membranes (Fig. 2B), we considered the possibility that a cytosolic factor(s) may be necessary to stimulate cargo recruitment into COPII vesicles. Indeed, the addition of a low level of cytosol stimulated the purified proteins to capture the cargo proteins p58 and APP (Fig. 5A, compare lanes 9–10 with lanes 13–14).



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  		FIG. 5.
Rat liver cytosol assists cargo packaging by recombinant COPII proteins. A and D, in vitro vesicle formation assay. Nucleotides represent an ATP regenerating system and 0.2 mM GTP. See "Experimental Procedures" for details. Ribophorin I is an ER resident protein that serves as a negative control, and p58 is an ERGIC protein that serves as a positive control. An aliquot (75%) of each vesicle fraction was loaded. B and C, GST pull-down assay. GST fusion proteins were incubated either with the Sec23A/24C complex or Sec23A alone. GST fusion proteins were captured using glutathione-agarose beads. After washing, the bound proteins were separated by SDS-PAGE. A region of the gel containing Sec23/24p was analyzed by immunoblotting (top panel), and a region of the gel containing the GST fusion proteins was stained with Coomassie Brilliant Blue R250 (bottom panel). Note that some background binding with GST was observed.

 



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  		FIG. 7.
APP and PS1 packaging are uncoupled. A, B, and D, in vitro vesicle formation assay. Rat liver cytosol (final 4 mg/ml) was used as a source of COPII. Nucleotides represent an ATP regenerating system and 0.2 mM GTP. A, microsomes from CHO-K1 cells were isolated as described under "Experimental Procedures." Where indicated, purified wild-type hamster Sar1a (WT) or GDP-restricted hamster Sar1a (T39N) was included in the reaction. B, microsomes from CHO-K1 cells were washed once with buffer G containing 1 M potassium acetate and with buffer G. C, quantification of B by 35S-labeled secondary antibody. D, microsomes were derived from PS1+/– fibroblasts, PS1–/– fibroblasts, PS +/+ embryonic stem cells, and PS–/–embryonic stem cells. r, (an ATP) regenerating system.

 
There are three possibilities to explain this synergy. First, an unknown factor(s) in the cytosol may be required for efficient COPII vesicle formation. Although the recombinant proteins were capable of generating COPII vesicles from liposomes, the vesicle-forming activity of the purified proteins may be further stimulated by this cytosolic factor(s). Second, the cytosolic factor(s) may enhance cargo loading. Thus, the purified proteins may make vesicles depleted of cargo. Third, the cytosolic factor(s) may be other Sec24 homologs (Sec24A, 24B, and 24D) because Sec24p has been implicated in most, if not all, cargo protein recognition (13, 14, 39–42). Purified Sec24C may not recognize p58 and APP, but small amounts of other Sec24 homologs present in the cytosol may cooperate with the recombinant proteins to make vesicles and select cargo molecules. The first two models invoke a non-COPII protein(s) required for efficient vesicle formation or cargo recruitment, whereas the third model suggests that other COPII subunits, very likely Sec24 homologs, are required for p58 and APP recruitment.

To test if Sec23A/24C recognizes p58 and APP, we fused the cytosolic portion of each cargo protein to GST. Published evidence suggests that the C-terminal cytoplasmic domain of p58 contains a diphenylalanine signal responsible for binding to Sec23/24 (41, 43). Purified GST fusion proteins were incubated with Sec23A/24C and sedimented using glutathione-agarose beads. Sec23A/Sec24C binding to GST fusion proteins was probed by immunoblotting for Sec23A and Sec24C. Interestingly, only GST-APPct interacted with Sec23A/24C (Fig. 5B). In contrast, Sec23A/24C did not interact with the cytoplasmic domain of rat Bet1 fused to GST (Fig. 5B), although the addition of hamster Sar1a and GMP-PNP promoted the formation of a Sec23A/24C/SNARE complex as we have shown for the yeast proteins (data not shown) (30). Sec23A alone did not support GST-APPct binding (Fig. 5C). Because the Sec24 subunit is responsible for cargo recognition, our results suggest that Sec24C is capable of recruiting APP but not p58. The Sec24 isozyme responsible for binding p58 has not been determined.

We examined the effect of excess Sec23A/24C on the activity of cytosol in packaging p58 and APP. If Sec24C-specific cargo molecules were preferentially packaged, the capture of other cargo may be diminished. Excess Sec13/31A had no inhibitory effect (Fig. 5D). The addition of Sec23A/24C without the other COPII proteins inhibited packaging of p58 but not of APP. This result is consistent with the direct interaction between GST-APPct and Sec24C (Fig. 5B). The stimulation of p58 packaging by cytosol and the COPII proteins may reflect a synergy of Sec24 isozymes in reactions where other coat proteins are not limiting. In this respect the C-terminal cytoplasmic domain of p58/ERGIC53 binds Sec24B in preference to Sec24C (41).

We fractionated the cytosol with ammonium sulfate to assess the distribution of COPII proteins and the budding/cargo stimulation activity. Most of the endogenous Sec23/24 and Sec13/31 complexes were precipitated at 30% ammonium sulfate (Fig. 6A), as reported by Aridor et al. (20). APP and p58 were packaged in reactions supplemented with this ammonium sulfate fraction (Fig. 6B, lanes 12 and 13). Although p58 was no longer packaged into vesicles by cytosol fractions above 30% ammonium sulfate, APP packaging was stimulated by cytosol fractions precipitated by up to 50% ammonium sulfate in an incubation supplemented with purified COPII proteins (Fig. 6, B and C, lanes 16–23). These results suggest that a non-COPII cytosolic factor(s) stimulates cargo packaging into vesicles. The factor that fractionates in the higher ammonium sulfate cut may be selective for packaging of APP. A comparable factor for p58 may co-fractionate with COPII protein in the 30% ammonium sulfate cut.



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  		FIG. 6.
Cytosol fractions devoid of endogenous Sec23/24 and Sec13/31 can still assist APP recruitment by recombinant COPII proteins. A, 0.05% of cytosol and serial ammonium sulfate cuts were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membrane strips were probed with the indicated antibodies. Note that the Sec24B antibody nonspecifically interacts with a cytosolic protein as marked by an arrowhead. B, in vitro vesicle formation assay. Nucleotides represent an ATP regenerating system and 0.2 mM GTP. C, quantification of B by 35S-labeled secondary antibody. r, (an ATP) regenerating system.

 
Using the in vitro vesicle formation assay, we monitored packaging of PS1. PS is endoproteolyzed to yield an N-terminal fragment (NTF) and a C-terminal fragment (44). Full length (FL) PS1 but not the N-terminal fragment was packaged into vesicles (Fig. 7B). Packaging of PS1 FL was inhibited by a dominant-negative form of hamster Sar1a, consistent with a role for COPII in this process (Fig. 7A). Our results suggest that PS1 FL is the substrate for transport by COPII vesicles and that endoproteolysis occurs in the vesicles or after PS1 is delivered to the ERGIC/Golgi. Processed PS1 is less abundant in the ER than in the Golgi complex in CHO cells (45). The PS1 N-terminal fragment may be retrieved to the ER and recycled to the Golgi at levels undetected by this budding reaction.

Cargo proteins are packaged into COPII vesicles at distinct threshold levels of Sar1p (46). Cargo proteins and their receptors may differ in their affinity for Sar1p and the sorting heterodimer, Sec23/24p. Although rat liver cytosol contains sufficient COPII protein to generate transport vesicles, Sar1 may be limiting as it is in yeast COPII vesicle-budding reactions driven by crude yeast cytosol. Packaging of endogenous PS1 FL increased in a Sar1a-dependent manner (Fig. 7A), reminiscent of the behavior of SNARE proteins in the yeast COPII budding reaction (46). In contrast, Sar1 levels in the cytosol were sufficient to package APP and p58 (Fig. 7A). Microsomal membranes washed with buffer containing 1 M potassium acetate displayed an enhanced dependence of PS1 FL on supplementation of cytosol with hamster Sar1a (Fig. 7, B and C), likely due to the removal of endogenous Sarl from the membranes. These results suggest that APP and PS1 FL are not coordinately packaged into COPII vesicles.

We further tested the packaging of APP using microsomes from PS1 null fibroblasts and PS1/PS2 double knock-out embryonic stem cells (Fig. 7D). APP packaging efficiency normalized to p58 in PS1+/– fibroblast cells (APP/p58 = 0.088 ± 0.001) was comparable with APP packaging efficiency in PS1–/– fibroblast cells (APP/p58 = 0.086 ± 0.01). Ablation of PS1 and PS2 did not reduce the packaging efficiency of APP (APP/p58 = 0.15 in ES PS+/+, APP/p58 = 0.25 in ES PS–/–). These results reinforce the idea that APP and PS1 export from the ER are not obligatorily linked.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report we introduce a biochemical reconstitution approach to examine an important first step in the traffic of membrane proteins implicated in familial forms of AD (FAD). PS1 and APP are compared with a standard marker of ER-to-Golgi traffic, p58, in a cell-free COPII vesicle budding reaction. The evidence suggests that both proteins are implicated in FAD traffic in COPII vesicles but that their transport may ordinarily be uncoupled. Furthermore, the results suggest that additional novel cytosolic proteins are required for the packaging of membrane proteins by the mammalian COPII proteins.

In many respects the mammalian COPII proteins resemble their yeast counterparts. The order of assembly of the proteins and their activity in a chemically defined liposome budding reaction are similar with the mammalian and yeast COPII ensemble. However, the purified mammalian proteins displayed only a background cargo packaging activity. The addition of crude rat liver cytosol to the pure COPII proteins restored the packaging of two different cargo proteins. Although ammonium sulfate fractionation suggests this cytosolic factor(s) is not a COPII subunit, we cannot be certain that the factor(s) stimulates vesicle budding or cargo sorting.

The cytosol requirement that we have observed with purified proteins was not detected in previous mammalian COPII vesicle formation reactions (19, 20). Two procedural differences may explain this discrepancy. First, our Sec13/31 was purified to homogeneity. Second, we separated vesicles without using trituration. The partially purified Sec13/31 used previously, or the application of a mechanical force in the budding reaction may have obscured the requirement for an additional factor. Our method was similar to the procedures we used to detect transport in the yeast system (22).

The cytoplasmic tail of p58 binds to Sec24B very effectively, whereas it binds to Sec24C with low affinity (41). However, we were not able to detect stable binding of Sec24C to p58. Sar1a did not activate Sec24C binding to p58, although it did potentiate an interaction with Bet1p (data not shown) (30). In addition, Sec24C did not package p58 into COPII vesicles (Fig. 6, B and C). These results suggest that Sec24C is not a cargo adaptor for p58. Direct interaction of APP with Sec24C and the ability of the recombinant COPII proteins to recruit APP strongly suggest that Sec24C serves as at least part of a cargo adaptor for APP. Sec24B in the 30% ammonium sulfate fraction or a different adaptor protein may serve to stimulate p58 capture.

The cytosolic factor could be a functional paralog of yeast Sec16p. Although yeast Sec16p is not essential for COPII vesicle formation in vitro, mammalian Sec16 may be required for productive vesicle formation in vitro (18). The soluble nature of this factor(s), however, is inconsistent with it being a Sec16p paralog, which is tightly associated with microsomal membranes in yeast (18). In addition, because the cytosolic activity is precipitated at rather high concentrations of ammonium sulfate (up to 50%, Fig. 6B), it is unlikely that the cytosolic factor is a large protein such as Sec16p. Hence, the cytosolic factor may be a novel protein with no equivalent in the yeast COPII system.

Purified yeast COPII proteins are sufficient to generate vesicles and package cargo molecules into vesicles from yeast microsomal membranes (22). In this respect, the mammalian reaction may be more complex. Protein exit from the ER is stalled during mitosis in vertebrate cells (47–49), a point of regulation that is not observed in yeast. Perhaps relevant to this regulation, protein kinase activity, lipid modification by phospholipase D, and O-glycosylation of Sec24 have been implicated in mammalian COPII vesiculation processes (34, 50, 51). We were not able to detect any molecular weight shift of Sec24C nor could we observe any effects of phospholipase D on the cargo capture activity of the recombinant proteins (data not shown). A protein kinase has been suggested to be responsible for the ATP requirement for COPII protein binding to microsomes (34). Because the ATP requirement is also seen with purified proteins and high salt- or urea-stripped membranes, the ATP-requiring molecule must reside in or firmly on the membrane. The cytosolic activity we detect may be a kinase but probably not the one proposed for stimulated COPII recruitment. Progress on the point requires purification of the additional activities implicated in APP and p58 packaging.

PS1 FL appears to be the principle PS1 species packaged into COPII vesicles. PS requires at least Nct, Aph-1, and Pen-2 for endoproteolysis and stability (for review, see Ref. 52). Although these four molecules constitute an enzymatically active {gamma}-secretase, proteolytic processing of PS is not a prerequisite for the enzymatic activity (44, 53, 54). The functional and intracellular location of this internal processing of PS1 remain obscure. At least one report suggested that PS1 processing can occur in the ER (55). However, our results suggest that PS1 exits the ER as an intact precursor. Considering that our budding reaction is incubated for an hour and that PS1 FL is not significantly converted to fragments, we prefer the idea that the processing occurs in distal compartments such as the ERGIC and cis-Golgi.

We find that packaging of APP and PS1 may be uncoupled (Fig. 7). However, we do not exclude the possibility that FAD-linked PS1 mutations disturb APP export. In fact, FAD-linked PS1 mutations are considered gain-of-function mutations. In addition, there is a report that some FAD-linked PS1 mutations affect APP export from ER (38). Nonetheless, our results suggest that most APP molecules are capable of exiting ER independently of PS1.


   FOOTNOTES
 
* The work was supported by funds provided by the Adler and Fidelity Foundations and the Howard Hughes Medical Institute (to R. S.) and the Swiss National Fund (to L. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720. Tel.: 510-642-5686; Fax: 510-642-7846; E-mail: schekman{at}berkeley.edu.

1 The abbreviations used are: PS, presenilin; AD, Alzheimer's disease; FAD, familial AD; APP, amyloid precursor protein; COPI, coat protein complex I; COPII, coat protein complex II; ER, endoplasmic reticulum; ERGIC, ER-to-Golgi Intermediate compartments; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; RT, room temperature; CHO, Chinese hamster ovary; GMP-PNP, guanosine 5'-({beta},{gamma}-imido)triphosphate; FL, full length. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Peter Espenshade (University of Texas Southwestern Medical Center, Dallas, TX), Hans-Peter Hauri (University of Basel, Basel, Switzerland), Wanjin Hong (Institute of Molecular and Cell Biology, Singapore, Singapore), Jean-Pierre Paccaud (Geneva University, Geneva, Switzerland), Rainer Pepperkok (EMBL Heidelberg, Germany), Sangram Sisodia (University of Chicago, Chicago, IL), Tom Rapoport (Harvard Medical School, Boston, MA), Jaakko Saraste (University of Bergen, Bergen, Norway), and Gopal Thinakaran (University of Chicago, Chicago, IL) for sharing valuable reagents. We acknowledge Marcus Lee for improving the manuscript and members of Schekman laboratory for discussion and encouragement, especially E. Futai and L. Miller. We also thank Ann Fisher in the cell culture facility for technical assistance.



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J. Kim, B. Kleizen, R. Choy, G. Thinakaran, S. S. Sisodia, and R. W. Schekman
Biogenesis of {gamma}-secretase early in the secretory pathway
J. Cell Biol., December 3, 2007; 179(5): 951 - 963.
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M. Garstka, B. Borchert, M. Al-Balushi, P. Praveen, N. Kuhl, I. Majoul, R. Duden, and S. Springer
Peptide-receptive Major Histocompatibility Complex Class I Molecules Cycle between Endoplasmic Reticulum and cis-Golgi in Wild-type Lymphocytes
J. Biol. Chem., October 19, 2007; 282(42): 30680 - 30690.
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H. J. Huttunen, S. Y. Guenette, C. Peach, C. Greco, W. Xia, D. Y. Kim, C. Barren, R. E. Tanzi, and D. M. Kovacs
HtrA2 Regulates beta-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation
J. Biol. Chem., September 21, 2007; 282(38): 28285 - 28295.
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I. Neeli, S. A. Siddiqi, S. Siddiqi, J. Mahan, W. S. Lagakos, B. Binas, T. Gheyi, J. Storch, and C. M. Mansbach II
Liver Fatty Acid-binding Protein Initiates Budding of Pre-chylomicron Transport Vesicles from Intestinal Endoplasmic Reticulum
J. Biol. Chem., June 22, 2007; 282(25): 17974 - 17984.
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M. Hiltunen, A. Lu, A. V. Thomas, D. M. Romano, M. Kim, P. B. Jones, Z. Xie, M. Z. Kounnas, S. L. Wagner, O. Berezovska, et al.
Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Abeta Secretion
J. Biol. Chem., October 27, 2006; 281(43): 32240 - 32253.
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D. Spasic, A. Tolia, K. Dillen, V. Baert, B. De Strooper, S. Vrijens, and W. Annaert
Presenilin-1 Maintains a Nine-Transmembrane Topology throughout the Secretory Pathway
J. Biol. Chem., September 8, 2006; 281(36): 26569 - 26577.
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