©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ras-related GTP-binding Protein, Rab1B, Regulates Early Steps in Exocytic Transport and Processing of -Amyloid Precursor Protein (*)

Jan M. Dugan (1), Christina deWit (2), Lisa McConlogue (2), William A. Maltese (1)(§)

From the (1) Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 and (2) Athena Neurosciences, South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The role of the Ras-related GTP-binding protein, Rab1B, in intracellular trafficking of -amyloid precursor protein (APP) was studied in cultured 293 cells. APP is processed via one of two alternative routes. In the major secretory pathway, APP is cleaved by -secretase within the region comprising the -amyloid peptide (A), resulting in release of a soluble NH-terminal exodomain (APP) and a 3-kDa peptide (p3) derived from the carboxyl-terminal tail. In the alternative amyloidogenic pathway, APP is cleaved by -secretase, with the release of a truncated exodomain (APP) and an intact A peptide. When APP was coexpressed with Rab1B(wt) or dominant-negative Rab1B mutants (Rab1B or Rab1B) there was a marked decrease in conversion of the immature Endo-H sensitive form of APP (108 kDa) to the mature O-glycosylated form of APP (130 kDa) in cells expressing the mutant forms of Rab1B. The block in Golgi-dependent processing of APP was accompanied by inhibition of secretion of APP (APP). A similar decrease in secretion of APP (APP + APP) was observed in cells that were coexpressing Rab1B with the ``Swedish'' variant of APP ( i.e. APP SW), which undergoes increased amyloidogenic processing. Coincident with the decline in APP secretion, the cells coexpressing APP SW with Rab1B showed a 90% decrease in A secretion. The data indicate that Rab1B plays a key role in endoplasmic reticulum Golgi transport of APP, and that APP must pass through a late Golgi compartment before entering either the -secretase or the amyloidogenic -secretase pathway. The results also suggest that mutant versions of other Rab proteins that function in different parts of the exocytic and endocytic pathways may be useful in defining the specific routes of APP transport involved in the biogenesis of A.


INTRODUCTION

The 4-kDa amyloid -peptide (A)() is a major component of cerebrovascular amyloid deposits associated with Alzheimer's disease (1, 2, 3) . A originates as a product of proteolytic processing of -amyloid precursor protein (APP), a ubiquitously expressed membrane glycoprotein that exists in three major isoforms (APP, APP, and APP) (2, 3, 4, 5, 6, 7) . The nascent or ``immature'' forms of APP undergo several modifications upon translocation from the endoplasmic reticulum (ER) to the Golgi apparatus. These include addition of O-linked oligosaccharides, tyrosine sulfation, and trimming of N-linked carbohydrates (8, 9, 10, 11) . APP is processed via one of two alternative proteolytic pathways (2, 3, 4) . In the major pathway, APP is cleaved within the A sequence by an endoprotease activity termed -secretase (12, 13, 14, 15) , releasing a 102-115-kDa amino-terminal exodomain which is secreted as a soluble protein, APP. The carboxyl-terminal remnant is then further degraded to yield a 3-kDa fragment (p3) which is also secreted (16, 17) . In the alternative processing pathway, APP is cleaved by an endoprotease activity termed -secretase in a manner that preserves the entire A sequence and releases a truncated exodomain, APP(18) . A is then generated after further proteolysis of the carboxyl-terminal stump (19, 20, 21, 22) .

Proteins belonging to the Rab subgroup of the Ras superfamily of small GTP-binding proteins have emerged as important regulatory components of the exocytic and endocytic vesicular transport pathways in mammalian cells (23, 24, 25, 26) . Individual members of the Rab family are uniquely localized in specific organelles and membrane compartments (27, 28, 29, 30, 31, 32, 33) . This suggests that different Rab proteins may control the directionality and/or specificity of particular steps in intracellular protein trafficking. Support for this concept has come from studies conducted with permeabilized cells and cell-free systems that reconstitute discrete steps in vesicular transport. Such assays have implicated Rab1B in ER Golgi transport (28) , Rab5 in early steps of endocytosis (34) , Rab8 in basolateral transport from the trans-Golgi network to the plasma membrane (33) , and Rab9 in transport from late endosomes to the trans-Golgi compartment (32) . The roles of specific Rab proteins have been further defined by studies in which overexpression of mutant Rab proteins with defective guanine nucleotide binding properties has been used to suppress the function of the corresponding endogenous Rab proteins in intact cells. For example, Tisdale et al.(35) showed that mutants of Rab1 or Rab2 inhibited ER Golgi transport of vesicular stomatitis virus glycoprotein in HeLa cells. Similar studies with Rab5 indicate a role for this protein in the endocytosis of transferrin (36) and horseradish peroxidase (37) in baby hamster kidney cells.

The targeting mechanisms that determine the alternative fates of APP remain to be defined, as do the exact subcellular sites of the various proteolytic processing events involved in the genesis of APP/p3 or APP/A. We hypothesize that many, if not all, of the steps in the intracellular trafficking of APP are mediated by distinct members of the Rab family. Consequently, functional perturbation of Rab proteins known to be localized in specific subcellular compartments may help to define the routes by which APP is directed to the alternative secretory or amyloidogenic processing pathways. As a first step in testing this approach, we have examined the effects of dominant-negative mutations in Rab1B on the posttranslational processing of APP. Herein we show that coexpression of GTP-binding-defective Rab1B mutants with APP in cultured human 293 cells inhibits the Golgi-dependent maturation of APP and decreases the secretion of APP. Mutations in Rab1B also inhibit secretion of A, suggesting that APP is transported at least as far as the medial Golgi compartment before entering the amyloidogenic processing pathway.


EXPERIMENTAL PROCEDURES

Mutagenesis of Rab1B

Mutations were introduced into the Rab1B sequence by means of overlap extension PCR (38) using pGEM3Z- Rab1B(39) as the template. The following 5` and 3` anchoring oligonucleotide primers were used in all PCR reactions: 5`-GTTGTAAAAC-GACGGCCAGTG and 5`-TCTGCTAGTGGTGGCTGCTGTTAGATAGGATCCCGT. The following mutator oligonucleotides and their complements were used to generate the indicated point mutations; 5`-GTGGGCAAGAATTGCCTGCTT (Rab1B) and 5`-CTGGTAGGCA-TCAAGAGTGAC (Rab1B). All PCR products were subcloned into pGEM3Z as EcoRI/ BamHI inserts. Rab1B proteins that were tagged by an amino-terminal myc epitope (EQKLISEEDL) were obtained by PCR modification of the wild-type or mutant Rab1B cDNAs, utilizing the following 5`- and 3`-oligonucleotide primers: 5`-GCCAGCGAATTCC-ATATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGAACCCCGA-ATATGACTAC and 5`-ACGGGATCCTATCTAACAGCAGCCACCACTAGCAGA. The resulting PCR products were digested with EcoRI and XbaI and subcloned into pCMV5neo (40, 41) to generate pCMV Rab1B, pCMV Rab1B, or pCMV Rab1B. The sequences of all Rab1B constructs generated by PCR modification were verified by the dideoxy chain termination technique using Sequenase 2.0 (U. S. Biochemical Corp.).

Construction of APP Expression Vectors

phCK751 and pohCK751sw were used for expression of wild-type or Swedish forms of APP, respectively. In both vectors the 655-bp fragment from HincII to AvaII of the cytomegalovirus immediate early gene promoter (CMV) drives expression of the NruI to SpeI fragment encoding human APP. The splice sequence positioned immediately 3` to the CMV promoter and 5` to the APP-encoding region was derived from a hybrid sequence of the adenovirus major late region first exon and intron and a synthetically generated IgG variable region splice acceptor. The 162-bp PvuII to HindIII fragment of the adenovirus major late region, containing 8 bp of the first exon and 145 bp of the first intron, was fused with a synthetically derived splice acceptor identical to the 99-bp HindIII to PstI fragment of the IgG variable region splice acceptor clone-6 (42) . A similar splice signal has been shown to enhance expression when placed 5` to the coding sequences (43) . Polyadenylation sequences are provided by the 884-bp HpaI to EcoRI fragment of SV40 containing the early polyadenylation signal. The BamHI site in this fragment was destroyed by mutagenesis. The PvuII to EcoRI fragment of pBR322 provides for phCK751 replication in Escherichia coli, with an engineered NotI site replacing the PvuII site. Herpes simplex type-1 viral replication and packaging sequences located between the plasmid and CMV sequences were derived from pON812 as described (44) . These sequences are not relevant to the experiments described in this report. The differences between phCK751 and pohCK751sw are in the presence of a familial Alzheimer's disease mutation and pUC-derived plasmid replication sequences in the latter vector. The mutation encodes a dual amino acid change (KM to NL) at positions 651 and 652 of APP(45) . In pohCK751sw, the NotI to PvuI fragment of phCK751 (containing the plasmid origin of replication) was replaced with the PvuII to PvuI fragment from pGEM3, thus allowing more efficient replication in E. coli.

Transient Expression of Rab1B and APP in Cultured Cells

Transformed human embryonic kidney cells (line 293), obtained from American Type Culture Collection, were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal calf serum. Cells were plated in 60-mm dishes at 1.8 10 cells/cm on the day before transfection. A calcium phosphate precipitation technique (46) was used to transfect cultures with phCK751 or pohCK751sw, alone or in combination with pCMV Rab1BWT, pCMV Rab1B, or pCMV Rab1B. After a 3-h exposure to the precipitated DNA, cells were subjected to glycerol shock (15% (v/v) glycerol in PBS), then fed with fresh DMEM with 10% serum. Transfection efficiencies and co-localization of transiently expressed proteins were monitored by dual-antibody immunofluorescence. Briefly, the transfected cells were fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 in PBS. After blocking with 5% powdered milk in PBS, cells were incubated with an affinity-purified rabbit polyclonal antibody (Anti-6) directed against residues 590-695 of human APP (10, 47) and the 9E10 monoclonal antibody against the Rab1B myc epitope tag. In parallel control reactions one or both primary antibodies were omitted. Bound primary antibodies were visualized with rhodamine B-conjugated goat anti-mouse IgG and fluorescence isothiocyanate-conjugated goat-anti rabbit IgG. A Nikon Optiphot-2 microscope was used with filter combinations EX546/DM580/BA590 and EX450-490/DM510/BA520 to distinguish red and green fluorescence, respectively.

Immunoblot Assays for Expression of APP , APP , and Rab1B

Washed cell pellets derived from 60-mm dishes were solubilized in sample buffer containing 8 M urea, 62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, and 5% 2-mercaptoethanol. For detection of APP, one-tenth of the total cell protein was subjected to SDS-PAGE on a 6.25% polyacrylamide gel (48) . For detection of Rab1B, one-fifth of the total protein was run on a 12.5% polyacrylamide gel. Proteins were transferred to Immobilon-P and the membranes were preincubated for 1 h in blotting solution (PBS containing 5% powdered milk and 0.1% Tween 20). Expression of Rab1B was monitored with monoclonal antibody 9E10 (Oncogene Science) directed against the c-Myc epitope tag on the Rab protein. Initially, the expression of APP was monitored with the Anti-6 polyclonal antibody, but in later studies (Figs. 5 and 6) a monoclonal antibody (8E5) with specificity against residues 444-592 of human APP (49) was used. The latter reagent was provided by Dr. Dale Schenk (Athena Neurosciences). All primary antibodies were used at a final concentration of 0.1 µg/ml, and incubations were carried out for 1 h. Secondary antibodies were either horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad), used at a 1:3000 dilution. Chemiluminescent detection was performed using the ECL kit (Amersham). In some experiments (Figs. 5 and 6), I-labeled goat anti-mouse IgG (0.45 µCi/ml; Du Pont NEN) was used as the secondary antibody to facilitate quantitation of the 8E5 monoclonal antibody bound to APP. After autoradiographic detection of bound I-labeled IgG, radioactivity was quantitated by placing sections of the blot in a -counter. The same immunoblotting procedure was used to quantitate the secreted exodomain derived from APP in samples of culture medium. Since the 8E5 antibody does not differentiate between APP and APP, the term APP is used to denote both possibilities.

Metabolic Labeling and Immunoprecipitation of APP and APP

Cell cultures were pulse-labeled for 10 min at 37 °C with 1 ml methionine-free DMEM containing 10% dialyzed fetal calf serum and 100 µCi of [S]methionine/cysteine (TranS-label, 1100-1200 Ci/mmol, ICN Inc.). The cells were then washed twice with PBS and subjected to a 40-min ``chase'' in medium containing 10% fetal calf serum, 2 mM methionine, and 2 mM cysteine. Cells from parallel cultures were harvested at the end of the pulse and chase periods and collected by centrifugation. Cell pellets were lysed in 500 µl of RIPA solution (100 mM Tris, pH 7.4, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Nonidet P-40) and lysates were cleared by centrifugation at 100,000 g for 20 min. The supernatant solution was incubated for 2 h at 4 °C with 5 µl of the 8E5 antibody, and immune complexes were collected by incubation for 1 h with protein A-Sepharose beads coupled with rabbit anti-mouse IgG. The beads were washed four times with RIPA, one time with PBS, and eluted by boiling for 10 min in 100 µl of SDS-PAGE sample buffer. Proteins were subjected to SDS-PAGE followed by fluorography (50) . The relative amounts of processed and unprocessed APP were determined from radioanalytic gel scans with an AMBIS 4000 detector. Endo-H sensitivity was determined by incubating the immunoprecipitated proteins overnight at room temperature with 10 milliunits of endoglycosidase H (Boehringer Mannheim) in 50 µl of 30 mM sodium citrate, pH 5.5, 0.75% SDS, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, prior to elution from the protein A-Sepharose beads (51) .

ELISA for A

The concentration of A was determined in samples of 48-h conditioned medium from transfected cultures by means of a sandwich-type ELISA, which employs a monoclonal antibody directed against an epitope in the A peptide that spans the site cleaved by -secretase and a second antibody directed against the first 16 amino acids of A (7) .


RESULTS

Generation of Rab1B Mutants

To begin to explore the relationship between Rab-mediated transport pathways and APP processing, two specific mutations that alter the GTP binding properties of Rab proteins and other Ras-related proteins were introduced into Rab1B. The first mutation entails substitution of Asn for Ser at position 22 (Rab1B). This Ser is analogous to Ser-17 in H-Ras, which is known to coordinate Mg and the phosphate of the guanine nucleotide (52) . Previous studies have indicated that this mutation greatly reduces the affinity of Rab1 proteins for GTP without altering their affinity for GDP (35, 53) . The second mutation entails substitution of Ile for Asn at position 121 (Rab1B). This Asn is equivalent to Asn-116 in the conserved NK XD sequence element in H-Ras, which is involved in binding the purine ring of GTP and GDP (54) . Rab1A and Rab1B proteins bearing this mutation fail to bind GTP and GDP in vitro(35, 55) . Both Rab1B and Rab1B behave as dominant-negative suppressors of endogenous Rab1 function when expressed in cultured HeLa cells, as evidenced by their ability to impair ER Golgi transport of vesicular stomatitis virus glycoprotein (35) . To facilitate quantitation of expressed Rab proteins by immunoblot assays, our wild-type and mutant Rab1B cDNAs were engineered to encode a Myc epitope tag at the amino-terminal end of the protein. Addition of such epitope tags to Ras-related proteins, including several Rab proteins, has not been found to alter their function or subcellular localization (53, 55, 56, 57, 58, 59) .

Coexpression of Rab1B with APP in 293 Cells

To test the effects of mutant Rab1B proteins on the intracellular transport and processing of APP, we developed a co-transfection/transient expression assay utilizing cultured human 293 cells. The wild-type and mutant Rab1B constructs described above were subcloned into the mammalian expression vector, pCMV5neo. The coding sequence for human APP was inserted into the vector, phCK751. When cells were transfected with DNA mixtures containing phCK751 combined with pCMV Rab1B, pCMV Rab1B, or pCMV Rab1B, expression of both the APP and Rab1B proteins was readily detected by immunoblot analysis of cell lysates after 24 h (Fig. 1). In cultures that were transfected with phCK751 alone, the Myc antibody gave no discernable signal in the 20-30-kDa region of the blot, which contains endogenous Rab proteins. Based on the results from a separate immunoblot performed with a polyclonal antibody directed against the carboxyl-terminal hypervariable region of Rab1B (not shown), the overall expression of Myc-tagged Rab1B in the transfected cultures was estimated to be 44 times that of the endogenous Rab1B. In accord with findings reported with other Rab proteins (60) , the expression of the Rab1B and Rab1B mutants was reduced by 55-75% compared to the wild-type protein (Fig. 1). However, these proteins were still expressed at levels that were 10-19-fold above that of endogenous Rab1B.


Figure 1: Immunoblots demonstrating coexpression of APP and Rab1B proteins in human embryonic kidney 293 cell cultures. Cells were transfected with 2 µg of phCK751, either alone or in combination with pCMV Rab1B (10 µg), pCMV Rab1B (30 µg), or pCMV Rab1B (30 µg). Cells were harvested 18 h after transfection and solubilized in SDS sample buffer. One-tenth of the total cellular protein was subjected to SDS-PAGE on a 6.25% polyacrylamide gel and immunoblotted with Anti-6 antibody against APP ( upper panel). One-fifth of the total protein was run on a 12.5% polyacrylamide gel and immunoblotted with 9E10 antibody against the myc-epitope on Rab1B ( lower panel). ECL was used for detection of bound IgG. Positions of molecular weight markers are indicated at the right of each panel.



APP was strongly overexpressed in 293 transfected with the phCK751 construct, regardless of whether or not pCMV Rab1B was cotransfected in the same culture (Fig. 1). Although 293 cells normally express some APP (10) , the endogenous APP was not detectable with the brief ECL exposures used to assay the transfected cells in this study. In the cultures that were transfected with phCK751 alone or in combination with pCMV Rab1B, the immunodetectable APP migrated on 12.5% SDS-polyacrylamide gels as a closely spaced doublet of approximately 108 and 130 kDa, corresponding to the unprocessed nascent polypeptide and the mature fully glycosylated protein, respectively (8, 10) . Although the nascent and mature forms of APP were poorly resolved in these initial studies of protein expression, it is noteworthy that in the cultures that had been cotransfected with plasmids encoding mutant Rab proteins ( i.e. Rab1B and Rab1B), there appeared to be a diminution in the 130-kDa band, with little or no change in the expression of the 108-kDa band. This observation provided the first hint that overexpression of the mutant Rab1B proteins might be affecting the posttranslational processing of APP.

Before proceeding with further analysis of APP processing in the cotransfected cells, an immunofluorescence study was conducted to determine the extent to which cotransfection resulted in the transient expression of both APP and Rab1B in the same population of cells. Cultures that had been transfected with phCK751 and pCMV Rab1B were analyzed by dual-antibody immunofluorescence. The overall transfection efficiency for the phCK751 + pCMV Rab1B combination was determined to be approximately 30%. Among the positive cells, 89% stained with both the anti-APP and anti-myc antibodies (data not shown). The staining with both antibodies exhibited a punctate distribution in discrete regions surrounding the nucleus, similar to the pattern recently described for Rab1B in NRK cells (61) . This is consistent with the expected localization of the majority of APP precursor and the Myc-tagged Rab1B in membranes of the ER and Golgi network. Transfection efficiencies and coexpression percentages similar to those just described were also seen in cultures that were transfected with phCK751 combined with the mutant pCMV Rab1B (data not shown).

Posttranslational Processing of APP Is Impaired in Cells Expressing Mutant Rab1B

Because of the high degree of overlap between cell populations overexpressing APP and Rab1B in the cotransfected cultures, it was feasible to conduct a pulse-chase study to determine whether the overexpression of wild-type or mutant Rab1B might have functional consequences for the posttranslational processing of APP. Cell-associated radiolabeled APP was immunoprecipitated from parallel transfected 293 cell cultures, either immediately after a 10-min pulse with [S]methionine, or after a 40-min chase to allow time for nascent APP to undergo ER Golgi transport and oligosaccharide maturation. As shown in Fig. 2A, when the immunoprecipitated APP was resolved by SDS-PAGE immediately after the pulse, the nascent 108-kDa form of APP was the only radiolabeled protein detected in all of the cultures. After the 40-min chase, significant accumulation of the mature 130-kDa form of APP could be seen in cells that were overexpressing APP, either alone or in combination with Rab1B. In contrast, the mature form of APP was almost undetectable in cells that were coexpressing APP with Rab1B. Instead, a poorly resolved APP band with a mobility slightly slower than that of the 108-kDa protein became visible after the 40-min chase (compare 0 versus 40 min lanes). The marked impairment of conversion of APP to the 130-kDa form in cells expressing Rab1B resembled the arrest of APP processing seen in cells treated with brefeldin A, an agent that is known to disrupt Golgi architecture and interfere with ER Golgi trafficking of glycoproteins (62, 63) (Fig. 2 A). Cultures in which APP was coexpressed with Rab1B (compared to Rab) showed a noticeable, but less severe, decrease in accumulation of radiolabeled 130-kDa APP after the 40-min chase (Fig. 2 A).


Figure 2: Pulse-chase analysis indicates that posttranslational processing of APP is impaired in cells expressing mutant Rab1B. 293 cells were cotransfected with phCK751 and the indicated pCMV Rab1B constructs as described in the legend to Fig. 1. Eighteen hours after transfection, parallel cultures were pulse-labeled for 10 min with [S]methionine and harvested immediately (0 min), or chased in medium with unlabeled methionine for 40 min. In one pair of cultures that had been transfected with phCK751, brefeldin A (2.5 µg/ml) was added to the medium 1 h prior to the pulse, and was maintained throughout the subsequent pulse-chase incubations. Cell pellets were solubilized and APP was immunoprecipitated as described under ``Experimental Procedures.'' Panel A shows the results of fluorography (48 h exposure) of dried SDS gels containing the immunoprecipitated APP from parallel cultures harvested before and after the 40-min chase. Panel B shows the results of two experiments in which the radioactivity in regions of the dried gel containing the processed (130 kDa) and unprocessed (108-115 kDa) forms of APP was used to calculate the ratio of processed to unprocessed protein at the end of the 40-min chase.



Because the levels of expression and labeling of nascent APP were somewhat different in each of the transfected cultures, fluorographic analysis was extended by direct quantitation of the radioactivity in gel segments containing the 130-kDa versus 108-115-kDa forms of APP at the end of the 40-min chase. When this ratio was used as an index of APP processing, it was clearly evident in two separate experiments that overexpression of Rab1B, and particularly Rab1B, resulted in substantial inhibition of the posttranslational maturation of APP (Fig. 2 B).

Low Molecular Mass Forms of APP Are Sensitive to Endoglycosidase H

Based on the localization of Rab1B in the ER, Golgi membranes, and transitional vesicles between these organelles (61) , it is reasonable to infer that the impaired maturation of APP observed in the foregoing pulse-chase study was a result of Rab1B or Rab1B interfering with Rab1-mediated transport steps required for delivery of nascent APP to the Golgi compartment. To further explore this possibility, cell-associated radiolabeled APP was immunoprecipitated from cotransfected cultures after a 1-h chase, and the precipitated protein was digested with Endo-H. The latter enzyme removes high-mannose N-linked oligosaccharides, which are present on newly synthesized glycoproteins in the ER, but does not cleave complex N-linked carbohydrates that have been trimmed by 1,2-mannosidase II and subjected to further modification ( e.g. addition of sialic acid) in the medial and late Golgi compartments (64) . As expected, the mature 130-kDa form of APP in cells expressing APP alone or in combination with Rab1B was insensitive to Endo-H (Fig. 3). In contrast, the 108-kDa form of APP isolated from all of the cultures, including those overexpressing the Rab1B and Rab1B, exhibited a mobility shift equivalent to approximately 2 kDa after treatment with Endo-H (Fig. 3). The magnitude of this mobility shift was identical to that previously reported when N-linked carbohydrate was removed from the nascent forms of APP by digestion with endoglycosidase F (10) . In the cells expressing Rab1B, the intermediate APP band (approximately 112 kDa), which was poorly resolved in the earlier pulse-chase study (see Fig. 2 A), was clearly separated from the 108-kDa form and also exhibited a mobility shift when exposed to Endo-H (Fig. 3). These findings support the conclusion that the lower molecular mass forms of APP, which accumulate in 293 cells expressing mutant Rab1B proteins, are localized in a pre-Golgi or early Golgi compartment.


Figure 3: Low molecular mass forms of APP in cultures expressing Rab1B mutants are sensitive to endoglycosidase H. Eighteen hours after transfection, 293 cell cultures expressing APP alone, or with the indicated Rab1B proteins, were pulse-labeled with [S]methionine for 10 min and chased for 40 min. At the end of the chase, APP was immunoprecipitated and incubated with (+) or without (-) Endo-H as described under ``Experimental Procedures.'' All samples were then subjected to SDS-PAGE and fluorography. The fluorograms shown in the figure were exposed for 40 h.



Secretion of APP Is Suppressed in Cells Expressing Mutant Rab1B

Endoproteolytic cleavage of APP by an enzymatic activity termed -secretase appears to occur in a late compartment of the secretory pathway, resulting in the release of the 120-kDa NH-terminal exodomain as a soluble protein, APP(3, 4) . Likewise, -secretase activity, which results in the production of APP and an amyloidogenic carboxyl-terminal tail, is thought to occur in a post-ER compartment. Therefore, impairment of protein trafficking between the ER and Golgi membranes might reduce the access of mature APP to the secretase enzymes and diminish the output of APP to the extracellular medium. To test this possibility, cells that were coexpressing APP with Rab1B or Rab1B were pulse-labeled with [S]methionine for 10 min to label nascent APP. The intracellular APP and the extracellular APP were then immunoprecipitated at the end of a 1-h chase. Because most of the APP generated from APP in 293 cells is the form, the results of this study reflect the activity of the -secretase pathway. As shown in Fig. 4, cells expressing Rab1B effectively processed APP to the mature 130-kDa form and secreted radiolabeled APP into the culture medium. Addition of brefeldin A to a parallel culture expressing Rab1B blocked the deposition of APP in the medium, confirming a previous report that trafficking of APP through an intact Golgi apparatus is essential for secretory processing (16) . When APP was coexpressed with Rab1B instead of Rab1B, intracellular processing of APP was markedly inhibited and accumulation of radiolabeled APP in the medium was undetectable after the 1-h chase (Fig. 4).


Figure 4: Secretion of APP is inhibited in cells coexpressing APP with Rab1B. Eighteen hours after transfection, 293 cell cultures that were coexpressing APP with either Rab1B or Rab1B were pulse-labeled for 10 min with [S]methionine. Cells were then chased for 1 h in medium containing 10% serum and both the cell monolayer and the conditioned medium were collected for immunoprecipitation with the 8E5 monoclonal antibody, which recognizes epitopes in APP and its secreted exodomain, APP. Where indicated, brefeldin A (2.5 µg/ml) was added to cells 1-h prior to the pulse, and maintained throughout the subsequent pulse and chase incubations. All immunoprecipitated proteins were subjected to SDS-PAGE and fluorography (72-h exposure). The radiolabeled APP immunoprecipitated from the cells before and after the 1-h chase is shown in the lanes marked Cells. The radiolabeled APP immunoprecipitated from the medium at the end of the chase is shown in the lanes marked Medium.



To further evaluate the effect of Rab1B expression on APP secretion, conditioned medium was obtained from cultures that were coexpressing APP with either Rab1B or Rab1B for 48 h. Aliquots of the medium were subjected to immunoblot analysis, using the 8E5 monoclonal antibody and a secondary goat anti-mouse I-labeled IgG to quantitate the relative amounts of accumulated APP in each sample. Data derived from two separate transfection studies indicated that coexpression of Rab1B with APP resulted in a marked reduction in the total APP that accumulated in the conditioned medium (Fig. 5 A). Similar results were obtained when the values for extracellular APP were normalized to the values for total intracellular APP in the cell monolayers, to compensate for variations in APP expression among the cultures (Fig. 5 B). In a separate study we determined that the amount of APP that accumulated in conditioned medium from cultures transfected with pCMV Rab1B alone was only 15-20% of that measured in cultures where pCMV Rab1B was cotransfected with phCK751. Thus, the small amount of APP in the medium from cultures coexpressing APP with Rab1B primarily reflects residual secretory processing of the expressed APP, rather than a background level of APP production from endogenous APP in the non-transfected cell population.


Figure 5: Accumulation of APP is reduced in medium from 293 cells coexpressing APP with Rab1B. Cells were cotransfected with phCK751 and either pCMV Rab1B or pCMV Rab1B. Immediately after transfection, cultures were fed with 2 ml of DMEM containing 10% fetal calf serum and were maintained in the same medium for 48 h. Both the conditioned medium and the cell monolayer were collected from each culture. Aliquots of conditioned medium (30-70 µl) were subjected to SDS-PAGE and immunoblot analysis, using I-labeled goat anti-mouse IgG to detect the 8E5 antibody bound to APP. The bound I-labeled IgG counts were used to determine the relative amount of accumulated APP in the 2 ml of conditioned medium. In parallel blots the total intracellular APP (both processed and unprocessed forms) was determined by the same method. For each culture the results were expressed as the total secreted APP in the medium ( Panel A), and as a normalized value obtained by forming a ratio between total secreted APP and total intracellular APP (cpm of bound I-labeled IgG) ( Panel B). The figure shows the results of two separate experiments, the first indicated by stippled bars and the second indicted by cross-hatched bars. Transfections were done in triplicate and each bar indicates the mean (± S.D.) of the determinations from three parallel cultures.



Production of A via Alternative Processing of APP Is Inhibited in Cells Expressing Rab1B

Since the foregoing studies indicated that Rab1B plays an essential role in early steps of trafficking of APP along the major -secretory pathway, we next asked what effect perturbation of Rab1B function might have on the processing of APP along the alternative -secretase pathway responsible for the biogenesis of A. To facilitate detection of A, these studies were conducted with cells that were transfected with the plasmid pohCK751sw, which encodes a variant form of APP found in a Swedish family exhibiting an autosomal dominant pattern of Alzheimer's disease. Although cells expressing the Swedish variant of APP continue to generate APP through the -secretase pathway, they exhibit substantially increased production of the -secretase product, APP, and the amyloid peptide, A (22, 65, 66) .

As in the case of wild-type APP, coexpression of Rab1B with the Swedish variant of APP ( i.e. APP SW) resulted in a 70-80% decrease in the accumulation of APP in conditioned medium from the transfected cultures (Fig. 6, A and B). This was true regardless of whether the results were expressed as total APP (Fig. 6 A) or a ratio of extracellular APP to intracellular APP (Fig. 6 B). Although it is likely that these results reflect a diminished production of both APP and APP, this cannot be stated definitively, since the antibody used in this study does not discriminate between the and forms of APP. A more direct measure of the activity of the amyloidogenic pathway in the transfected cells was obtained by using an ELISA to quantitate the level of A in samples of conditioned medium from the same cultures used for the APP assays. The results indicate that the decline in production of APP in cultures expressing Rab1B was accompanied by a parallel decrease in the secretion of A into the medium (Fig. 6, C and D). Thus, both the amyloidogenic and secretory processing of APP were clearly affected by perturbation of Rab1B-mediated trafficking events. In fact, the data suggest that interference with the function of Rab1B may have an even greater impact on the events leading to A release than it is does on the secretion of APP. This point is underscored by a comparison of the A/APP ratios in samples of medium derived from cultures that were coexpressing APP SW with either Rab1B or Rab1B (Fig. 6 E). At present, the mechanism underlying the apparent differential inhibition of A versus APP biogenesis in cells expressing Rab1B remains to be defined.


Figure 6: Accumulation of both A and APP is reduced in medium from 293 cells coexpressing an amyloidogenic variant of APP with Rab1B. Cells were cotransfected with pohCK751sw and either pCMV Rab1B or pCMV Rab1B. Immediately after transfection, cultures were fed with 2 ml of DMEM containing 10% fetal calf serum and were maintained in the same medium for 48 h. Both the conditioned medium and the cell monolayer were collected from each culture. Aliquots of conditioned medium and cell lysate were subjected to SDS-PAGE and immunoblot analysis to determine the relative amounts of secreted APP and intracellular APP in the cultures. The concentration of A was determined by ELISA in aliquots of conditioned medium from the same cultures. The results of the APP assays were expressed as total secreted APP (based on cpm of bound I-labeled IgG on the immunoblots) per 2 ml of conditioned medium ( Panel A) and as a ratio of total secreted APP (cpm) to total immunodetectable cellular APP (cpm) for each culture ( Panel B). The results of the A assays were expressed as the total A (ng) accumulated in 2 ml of conditioned medium ( Panel C) and as nanograms of extracellular A per unit of cellular APP (1 unit = 10 cpm of bound I-labeled IgG) ( Panel D). The ratio of total A (ng) to total immunodetectable units of APP (1 unit = 10 cpm bound I-labeled IgG) in the conditioned medium from each culture is depicted in Panel E. Transfections were done in triplicate and each bar indicates the mean (± S.D.) of the determinations from three parallel cultures.




DISCUSSION

Previous studies by Balch and co-workers (35, 53, 55) have established that Rab1A and Rab1B mutants that are defective in GTP-binding can block ER Golgi transport and processing of virus-encoded glycoprotein when expressed in mammalian cells infected with vesicular stomatitis virus. These dominant-negative effects have suggested that Rab1 proteins may play a global role in ER Golgi transport of a variety of other integral membrane glycoproteins that undergo oligosaccharide maturation and/or O-glycosylation as they traverse the Golgi compartment en route to the cell surface. The present demonstration that Rab1B, and to a lesser extent Rab1B, can impair the conversion of Endo H-sensitive precursor forms of APP to the higher molecular weight O-glycosylated forms, provides strong independent evidence to support this view.

Although both Rab1B and Rab1B inhibited the conversion of APP to the fully glycosylated 130-kDa form, the effect of Rab1B was much more pronounced than the effect of Rab1B (see Fig. 2). Recent studies focusing on Rab1A, a functionally interchangeable isoform of Rab1B, indicate that these two mutations may interfere with the physiological function of Rab1 proteins through different mechanisms. The S25N mutation in Rab1A reduces the affinity of the protein for GTP without altering its affinity for GDP, thus favoring retention of the protein in the GDP state (53) . In contrast, the N124I substitution in Rab1A (equivalent to N121I in Rab1B) decreases the affinity of Rab1A for both GTP and GDP (55) . Morphological analyses suggest that the inhibition of vesicular stomatitis virus glycoprotein transport by Rab1A or Rab1B occurs primarily at the level of vesicle budding from the ER (53) , whereas the block in ER Golgi transport produced by Rab1A or Rab1B occurs as a result of interference with the targeting or fusion of transitional vesicles with the cis-Golgi (35, 55) . Interestingly, both mutants have been shown to cause dispersion of the Golgi apparatus when they are microinjected into rat fibroblasts, possibly indicating a disruption of the equilibrium between anterograde and retrograde transport through this organelle (67) . The latter observation is consistent with the hypothesis that in addition to regulating ER Golgi transport, Rab1B may play a role in regulating early steps in intra-Golgi vesicle trafficking (28) .

As mentioned earlier, the initial endoproteolytic cleavage of APP by -secretase is believed to occur after the protein has traversed the trans-Golgi compartment and undergone O-glycosylation and tyrosine sulfation (13, 20, 68, 69) . The studies described in Figs. 4 and 5 confirm this model by showing that the decreased flux of APP into the Golgi compartment in cells coexpressing APP with Rab1B is accompanied by a substantial reduction in secretion of APP. It is noteworthy that the inhibition of secretion of APP in cells expressing Rab1B was not complete; i.e. the cumulative levels of APP in samples of 48-h conditioned medium were approximately 30% of those detected in cultures expressing Rab1B. There are several possible explanations for this finding. The most obvious possibility is that the block in ER Golgi transport mediated by Rab1B is leaky, so that over an extended period of time enough APP might reach the trans-Golgi network and plasma membrane to generate detectable quantities of secreted APP. An alternative possibility is that a small portion of the relatively large intracellular pool of immature APP in transfected 293 cells is subject to endoproteolytic cleavage by an unidentified enzyme in the ER, generating an amino-terminal APP fragment that is recognized by the 8E5 antibody. If the resulting APP fragment could be translocated to the cell surface without traversing the Golgi apparatus, one might expect its extracellular accumulation to be insensitive to the block in ER Golgi trafficking by Rab1B. In this regard, Gabuzda et al.(70) have recently described a protease activity that degrades the immature form of APP in COS cells, yielding an 11.5-kDa peptide that contains the intact A sequence. It is not yet known whether this activity results in the release of an intact amino-terminal exodomain.

The experiments conducted with the Swedish variant of APP ( i.e. APP SW) provide new information about the pathway for generation of A. When Rab1B was coexpressed with APP SW, the secretion of APP was suppressed to the same extent as in the experiments with wild-type APP. Since the APP derived from APP SW is a mixture of APP and APP, whereas the APP derived from wild-type APP is predominantly APP(65) ,() these findings suggest that transport of APP from the ER to the Golgi apparatus is required for entry of the protein into the -secretase pathway as well as the -secretase pathway. This conclusion is strengthened by the finding that the concentration of A was markedly reduced in conditioned medium from cells that were coexpressing APP SW with Rab1B. The foregoing observations are entirely consistent with previous studies in which both APP maturation and A production were inhibited by brefeldin A (16) . The results also indicate that if immature APP can undergo proteolysis in the ER or early Golgi to form potentially amyloidogenic carboxyl-terminal fragments, as suggested in a recent study (70) , then Rab1B-mediated transport events may be required for translocation of these fragments to a compartment where they can be further processed to yield A.

The differential decline in A versus APP in conditioned medium from cells coexpressing APP SW with Rab1B instead of Rab1B (see Fig. 6 E) was unexpected, and raises the intriguing possibility that interference with Rab1B-mediated export of APP from the ER has a more severe impact on amyloidogenic processing of APP than it does on the -secretase processing of the protein. At present we can only speculate about the types of mechanisms that might underlie this phenomenon. One possibility is that the -secretase has a higher Kfor mature APP substrate than does the -secretase, so that even a modest decline in the pool of mature APP traversing the Golgi compartment has a substantial impact on -cleavage and A production, while allowing some -cleavage to continue. Alternatively, it is possible that the concentration of mature APP residing in a specific compartment ( e.g. the trans-Golgi network) must exceed a particular threshold value before the steps involved in A production (-secretase cleavage and possible endosomal processing of the carboxyl-terminal domain) can operate at optimal efficiency. The latter model would be consistent with a recent study in which amino acid substitutions that impair cleavage of APP at the -secretase site were shown to increase A production, possibly by increasing substrate availability for the -secretase pathway (71) . Future studies using antibodies that can discriminate between the APP and APP secretory products should help to distinguish between these potential mechanisms.

Although this study focuses on well defined early steps in the secretory processing of APP, it should be emphasized that the transport routes which determine alternative amyloidogenic processing of APP remain poorly characterized. For example, while acidic compartments such as the endosome or lysosome have been implicated in the production of A (16, 72, 73) , it is uncertain whether entry of APP and its carboxyl-terminal remnants into this pathway occurs via internalization from the plasma membrane, translocation from an intracellular compartment such as the trans-Golgi network, or both. Thus, aside from documenting a role for Rab1B in ER Golgi transport of APP, the results of the present study clearly support the feasibility of using dominant-negative mutations in additional members of the Rab protein family to define and distinguish other lesser known segments of the APP processing routes. This experimental approach provides a powerful new tool for dissection of this critical pathway in the pathogenesis of Alzheimer's disease.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA34569 (to W. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2616. Tel.: 717-271-6675; Fax: 717-271-6701; E-mail: wmaltese@geisinger.edu.

The abbreviations used are: A, amyloid -peptide; APP, -amyloid precursor protein; APP, soluble NH-terminal exodomain derived from APP; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Endo-H, endoglycosidase H; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; bp; base pair(s); CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay.

L. McConlogue and W. A. Maltese, unpublished data.


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