©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Effects of a Rab6 Mutant on Secretory Versus Amyloidogenic Processing of Alzheimers -Amyloid Precursor Protein (*)

(Received for publication, October 19, 1995)

Lisa McConlogue (1)(§) Flavia Castellano (2)(§) Christina deWit (1) Dale Schenk (1) William A. Maltese (2)(¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Ras-related GTP-binding protein, Rab6, is localized in late Golgi compartments where it mediates intra-Golgi vesicular trafficking. Herein we report that coexpression of Alzheimer's beta-amyloid precursor protein (betaAPP) with a dominant-negative Rab6 mutant (Rab6) in human embryonal kidney 293 cells causes an increase in secretion of the soluble amino-terminal exodomain (s-APPalpha) derived from non-amyloidogenic processing of beta-APP by alpha-secretase. The effect was specific to Rab6, since the corresponding mutation in Rab8 (i.e. Rab8), which has been implicated in protein transport to the plasma membrane, caused a modest reduction in s-APPalpha secretion. While Rab6 stimulated secretion of APPalpha, the accumulation of amyloid beta peptide (Abeta) in the medium was either moderately reduced or unaffected. Similar differential effects of Rab6 on secretion of s-APPalpha versus Abeta were observed in cell cultures that were overproducing Abeta after transfection with a plasmid encoding the Swedish variant of betaAPP. Moreover, assays of medium from the latter cultures revealed a marked increase in secretion of s-APPalpha relative to s-APPbeta (the immediate product derived from cleavage of betaAPP by beta-secretase). The results indicate that vesicular transport events controlled by Rab6 occur at or near a critical juncture in the trans-Golgi network where betaAPP is sorted into either the constitutive alpha-secretase pathway or the amyloidogenic beta-secretase pathway.


INTRODUCTION

The 4-kDa amyloid beta-peptide (Abeta) (^1)has been implicated in the pathogenesis of Alzheimer's disease(1, 2) . Abeta is formed as the result of intracellular proteolytic processing of beta-amyloid precursor proteins (betaAPP, betaAPP, and betaAPP). The biogenesis of Abeta begins when betaAPP is cleaved by an enzymatic activity termed beta-secretase, releasing a soluble NH(2)-terminal exodomain (s-APPbeta), which is secreted from the cell, and leaving a membrane-anchored COOH-terminal fragment containing the intact Abeta sequence(3, 4, 5, 6) . Abeta is released when the latter fragment is further trimmed by another proteolytic activity currently referred to as -secretase(7, 8) . While amyloidogenic processing of betaAPP is known to occur at low levels ubiquitously, cells that produce betaAPP direct the substantial proportion of the precursor protein to the constitutive secretory pathway, where it is processed via a non-amyloidogenic mechanism. The latter entails initial cleavage of betaAPP within the Abeta domain by an enzymatic activity termed alpha-secretase, releasing a soluble exodomain (s-APPalpha) containing part of the Abeta sequence(3, 9, 10, 11) . Because the residual COOH-terminal stump lacks an intact Abeta domain, it cannot give rise to Abeta when cleaved by -secretase.

The mechanisms by which cells control the flux of betaAPP into the amyloidogenic versus non-amyloidogenic pathways are poorly understood. It has been particularly difficult to determine the precise subcellular sites of the various processing events because the relevant proteases have not yet been isolated. However, there is sufficient evidence to suggest that alpha-secretase acts on the mature form of betaAPP at the cell surface or in a late compartment of the default secretory pathway, after it has undergone tyrosine sulfation(10, 12, 13, 14, 15) . Less is known about the subcellular sites of the amyloidogenic processing of betaAPP by beta-secretase and -secretase. A number of studies have indicated that events occurring in acidic compartments (e.g. endosomes or vesicles of the trans-Golgi network) are involved in the biogenesis of Abeta(16, 17, 18, 19) . In particular, a recent study suggests that beta-secretase cleavage of betaAPP containing the Swedish mutation occurs within transitional vesicles between the trans-Golgi compartment and the cell surface(20) .

To elucidate the steps involved in intracellular trafficking and processing of betaAPP, we have adopted a novel strategy, which is based on mutagenesis and expression of GTP-binding proteins encoded by the rab gene family. There are currently more than 30 distinct Rab proteins, which are localized in discrete organelles and vesicles, where they play key roles in protein trafficking between specific donor and acceptor compartments along the exocytic or endocytic routes (21, 22, 23) . Like other Ras-related proteins, Rab proteins are active when they are in the GTP-bound state. Hence, mutant Rab proteins with reduced affinity for GTP exert a dominant negative effect over their endogenous counterparts when overexpressed in mammalian cells(24, 25, 26) . We recently showed that when betaAPP was coexpressed with a dominant-negative Rab1B mutant (i.e. Rab1B) in HEK 293 cells, the maturation of betaAPP and the secretion of both s-APP and Abeta were impaired(27) , consistent with the established role of Rab1B in ER Golgi transport(24, 28, 29) .

In the present study we focused on Rab6, which is known to reside in trans-Golgi cisternae, the trans-Golgi network, and post-Golgi secretory vesicles(30, 31, 32) , and has been implicated in both intra-Golgi transport (33) and the budding of exocytic vesicles from the trans-Golgi network(34) . We found that when betaAPP was coexpressed with a GTP-binding-defective Rab6 mutant (Rab6), secretion of s-APPalpha was stimulated, while deposition of Abeta and s-APPbeta into the medium was either modestly reduced or unaffected. These findings suggest that transport steps mediated by Rab6 occur at a branch point where amyloidogenic and non-amyloidogenic pathways for betaAPP processing diverge.


EXPERIMENTAL PROCEDURES

Mutagenesis of Rab6 and Rab8

The cDNAs encoding human Rab6 (35) and Rab8 (36) were obtained by PCR amplification from first strand cDNA template, reverse-transcribed from total Hela cell mRNA(37) . Each construct was further modified by PCR to encode a 10-amino acid Myc epitope tag at the amino terminus of the expressed protein, as described previously for Rab1B(27) . The myc-rab6 cDNA was ligated into the EcoRI/BamHI sites of the mammalian expression vector pCMV5(38) , to obtain pCMVrab6. The myc-rab8 cDNA was ligated into the EcoRI/XbaI sites of pCMV5neo (39) to obtain pCMVrab8. The latter vectors were used as templates to generate DNA's encoding Myc-Rab6 and Myc-Rab8 by site-directed mutagenesis, using the PCR-based megaprimer method (40) with the following mutator oligonucleotides: Rab6, 5`-CTAGTAGGAATCAAAACAGATCTTGCTGAC; Rab8, 5`-CTTGGGATCAAGTGTGATGTG. The resulting PCR products were subcloned into pCMV5 to obtain pCMVrab6 and pCMVrab8. The DNA sequences of all constructs were verified by the dideoxy chain termination technique using the Sequenase 2.0 kit (United States Biochemical Corp.).

Coexpression of betaAPP with Rab Proteins

Expression vectors encoding betaAPP (phCK751) and the Swedish variant of betaAPP (pohCK751sw) have been described previously(27) . Human 293 cells were grown in 60-mm dishes and transfected exactly as described by Dugan et al.(27) . For transient coexpression of betaAPP (WT or Swedish) with Rab6 (WT or N126I) or Rab8 (WT or N121I) in 293 cells, 10 µg of the specified rab pCMV DNA was combined with 1 µg of phCK751 or pohCK751sw.

Immunoblot Analyses

For analysis of intracellular proteins, washed cell pellets derived from 60-mm dishes were solubilized in SDS sample buffer and subjected to SDS-PAGE and immunoblot analysis as described previously(41, 42) . To confirm expression of the Rab proteins, one fourth of the cell lysate was subjected to SDS-PAGE on a 12.5% polyacrylamide gel and immunoblotted with 9E10 monoclonal antibody against the Myc epitope tag (Oncogene Sciences) or polyclonal affinity-purified rabbit antibodies directed against the hypervariable regions of Rab6 or Rab8 (Santa Cruz Biotechnology). To quantitate intracellular betaAPP (both mature and immature forms), one fourth of the cell lysate was subjected to SDS-PAGE on a 6% polyacrylamide gel and immunoblotted with the 8E5 monoclonal antibody with specificity against residues 444-592 of human betaAPP(43) . To quantitate total secreted s-APP in individual cultures, 50 µl of the conditioned culture medium (out of 2 ml total) was subjected to SDS-PAGE and immunoblotted with the 8E5 antibody. In experiments involving expression of the Swedish variant of betaAPP (SWbetaAPP), the alpha and beta forms of s-APP were distinguished by using the SW192 polyclonal antibody (18) to detect s-APPbeta and the 2H3 monoclonal antibody, which is directed against residues 1-12 of the Abeta sequence, to detect s-APPalpha. The latter reagent was provided by Grace Gorden, Athena Neurosciences. To facilitate quantitation of bound primary IgG, I-labeled secondary antibodies were used (i.e. rabbit anti-mouse or goat anti-rabbit IgG) and blots were either counted in a counter or scanned with a Molecular Dynamics PhosphorImager.

ELISA for Abeta and Abeta

Samples of conditioned culture medium were cleared of s-APP forms, which can interfere with the Abeta ELISA, by absorption with heparin-agarose resin as follows. Heparin-agarose (H-6508, Sigma) was washed in Tris-buffered saline (Tris-HCl, pH 7.5, 0.15 M NaCl) three times and resuspended in an equal volume of Tris-buffered saline. 100 µl of the heparin-agarose slurry was added to 1 ml of culture medium and incubated overnight at 4 °C on a rocker. The samples were centrifuged at 14,000 rpm in a microcentrifuge for 5 min, and the resulting supernatant solutions were analyzed for Abeta forms by specific ELISA. The concentration of total Abeta was determined by means of an ELISA employing capture by monoclonal antibody 266, directed against an epitope in the Abeta peptide that spans the site cleaved by alpha-secretase, and detection with the monoclonal antibody 6C6, which is directed against the first 16 amino acids of Abeta(44) . The ELISA used for determination of the concentration of Abeta also employed monoclonal antibody 266 for capture, but used for detection a polyclonal antibody 277-2, which was raised against a peptide including Abeta residues 33-42(45) .


RESULTS

We recently showed that the inhibitory effects of a Rab1B mutation (N121I) on early steps in the posttranslational maturation of betaAPP can be readily detected by transiently coexpressing both proteins in HEK 293 cells(27) . In the present study, we used the same approach to examine the possible role of a Golgi-localized Rab protein, Rab6, in the secretory and amyloidogenic processing of betaAPP. By analogy to other members of the Ras superfamily(46, 47) , incorporation of the N126I substitution into Rab6 is predicted to drastically reduce the affinity of the protein for GTP. Accordingly, Myc-Rab6 immunoprecipitated from 293 cells with anti-Myc antibody failed to bind detectable [P]GTP when compared to Myc-Rab6 in a standard blot-overlay assay (not shown). To determine the effects of this Rab6 mutation on secretion of s-APP and Abeta in 293 cells, Myc-Rab6 and Myc-Rab6 were each coexpressed with betaAPP for 48 h and the relative amounts of s-APP and Abeta were measured in samples of conditioned medium (Fig. 1). To exclude the possibility that differences in extracellular s-APP or Abeta might reflect variations in betaAPP expression in the cell monolayers, the values for s-APP and Abeta were normalized to the total intracellular betaAPP in each culture. Compared to cultures that were not transfected with Rab6 plasmid, the cultures that were coexpressing Rab6 with betaAPP showed a modest reduction in secreted s-APP. The opposite effect was observed in cultures expressing Rab6, where levels of s-APP were markedly elevated (Fig. 1A). In contrast to the level of s-APP, the extracellular concentration of Abeta did not increase in the cultures that were expressing Rab6 (Fig. 1B). In fact, the Abeta levels were lower in these cultures than in the cultures expressing Rab6 or no exogenous Rab6. The inverse effects of Rab6 on s-APP and Abeta in this study are underscored by the 5-fold decline in the direct ratio of extracellular Abeta to extracellular s-APP in the cultures expressing Rab6versus Rab6 (Fig. 1C).


Figure 1: Rab6 has differential effects on secretion of s-APP versus Abeta. Cells were cotransfected with phCK751 alone or in combination with plasmids encoding Myc-tagged Rab6 or Rab6, as indicated. All cultures were incubated without changing the medium for 48 h. Expression levels of the Myc-Rab6 or Myc-Rab6 ranged from 3- to 5-fold over the endogenous Rab6, as determined by immunoblot analysis with an affinity-purified antibody against Rab6 (not shown). Expression levels of betaAPP were approximately 40-fold over the endogenous betaAPP in 293 cells, so that measurements of products derived from betaAPP (i.e. s-APP) reflect almost exclusively the processing of the overexpressed betaAPP in the subpopulation of cells expressing the Rab6 constructs. Upon harvesting the cultures, levels of s-APP (counts/min) and Abeta (picograms) were determined in aliquots of the conditioned medium by Western blot assay using IgG or ELISA, respectively (see ``Experimental Procedures''). The values for total s-APP (panel A) and Abeta (panel B) in 2 ml of medium were normalized to the total intracellular betaAPP in the cell monolayer. In panel C, the values for total extracellular Abeta were expressed as a ratio to total extracellular s-APP. The results shown are means (± S.E.) of separate determinations performed on three parallel cultures.



To verify the specificity of the results observed with the Rab6 mutant, the equivalent mutation (N121I) was introduced into Rab8. The latter protein is localized to the cell periphery (48, 49) and is normally expressed in a wide variety of cells, including 293 cells. (^2)Rab8 has been implicated as a mediator of protein transport from the trans-Golgi network to the basolateral (50) or dendritic (51) plasma membrane in polarized epithelial and neuronal cells, respectively. However, it is unclear whether Rab8 plays a similar role in non-polarized cells. When betaAPP was coexpressed with Rab8, the relative amount of secreted s-APP was modestly reduced in comparison to parallel cultures expressing Rab8 (Fig. 2A). The levels of Abeta were also slightly reduced in the cultures expressing Rab8 (Fig. 2B), and there was no change in the direct ratio of Abeta to s-APP in the culture medium (Fig. 2C). These findings contrast with the striking decrease in the ratio of Abeta/s-APP (mostly due to increased secretion of s-APP) observed in the cultures expressing Rab6 (Fig. 1C).


Figure 2: Rab8 affects s-APP secretion in a manner distinct from Rab6. Cells were cotransfected with phCK751 alone or in combination with plasmids encoding Myc-tagged Rab8 or Rab8, essentially as described in the studies with Rab6. All cultures were incubated without changing the medium for 48 h. Upon harvesting the cultures, levels of s-APP (counts/min) and Abeta (picograms) were determined in aliquots of the conditioned medium by Western blot assay using IgG or ELISA, respectively (see ``Experimental Procedures''). The values for total s-APP (panel A) and Abeta (panel B) in 2 ml of medium were normalized to the total intracellular betaAPP in the cell monolayer. In panel C, the values for total extracellular Abeta were expressed as a direct ratio to total extracellular s-APP. The results shown are means (± S.E.) of separate determinations performed on three parallel cultures.



To determine whether the higher end point values for accumulated extracellular s-APP in cultures expressing Rab6 reflected an increased rate of secretion of s-APP, samples of medium from individual cultures overexpressing betaAPP with either Rab6 or Rab6 were collected at multiple time points over a 24-h period (Fig. 3A). The results of this analysis demonstrated an increased rate of s-APP accumulation in the cultures expressing Rab6 compared to cultures expressing Rab6. The increased deposition of s-APP into the medium could not be attributed to differential expression of betaAPP, since the intracellular levels of mature (fully glycosylated) betaAPP (Fig. 3B) and immature betaAPP (Fig. 3C) were comparable in cultures expressing both the wild-type and mutant Rab6 proteins. Consistent with the results obtained in the 48-h coexpression experiment (Fig. 1), the 24-h end point values for extracellular s-APP, normalized to total intracellular betaAPP, were increased approximately 2-fold in the cultures expressing Rab6versus Rab6 (Fig. 3D), while the values for Abeta were not increased (Fig. 3E).


Figure 3: Rate of secretion of s-APP is increased in cells coexpressing betaAPP with Rab6versus Rab6. Beginning 18 h after transfection, fresh medium (2 ml) was added to each culture and 50-µl aliquots were removed at the designated intervals for quantitation of total s-APP (panel A). After the last samples were removed, the cell monolayers were harvested and the intracellular levels of mature betaAPP (125-130 kDa) (panel B) and immature betaAPP (108-110 kDa) (panel C) were determined. The values for total s-APP (panel D) and Abeta (panel E) in the conditioned medium at 24-h are also expressed as normalized values (ratio to the total intracellular betaAPP). Values for s-APP and betaAPP are derived from Western blot assays employing I-IgG and are expressed as total phosphorimager units/culture. Values for total Abeta (picograms) are derived from ELISA. Each value is a mean (± S.E.) of determinations performed on three parallel cultures.



The s-APP secreted by 293 cells expressing wild-type betaAPP is predominantly the alpha form (i.e. s-APPalpha) released upon cleavage by alpha-secretase(52) . In light of the contrasting effects of Rab6 on levels of s-APP and Abeta, we were interested in determining how expression of this Rab6 mutant might affect secretion of the immediate product generated by beta-secretase cleavage (i.e. s-APPbeta). To facilitate this analysis, 293 cells were cotransfected with plasmids encoding either Rab6 or Rab6 together with the Swedish variant of betaAPP (i.e. SWbetaAPP), which contains a dual amino acid change (Lys Asn/Met Leu) known to promote increased processing of the protein along the Abeta pathway (52, 53, 54) . Using antibodies that discriminate between alpha and beta forms of s-APP, we observed that the amount of s-APPalpha (normalized to intracellular SWbetaAPP) secreted into the medium was almost doubled in cultures expressing Rab6 (Fig. 4A). In contrast, the normalized value for extracellular s-APPbeta was modestly reduced in the same cultures (Fig. 4B). These reciprocal changes were reflected in a 3-fold increase in the direct ratio of the alpha and beta forms of s-APP in the medium from cultures coexpressing SWbetaAPP with Rab6versus Rab6 (Fig. 4C). Although Rab6 caused a small decline in the level of extracellular s-APPbeta in this experiment, we were unable to detect a significant decrease in intracellular s-APPbeta (referred to as c-APPbeta in Fig. 4D). This suggests that expression of Rab6 does not interfere directly with transport steps required for SWbetaAPP to gain access to the subcellular compartment containing beta-secretase.


Figure 4: Differential changes in s-APPalpha versus s-APPbeta and Abeta in 293 cells coexpressing the Swedish variant of betaAPP with Rab6. Cells were cotransfected with pohCK751sw alone or in combination with plasmids encoding Myc-tagged Rab6 or Rab6, as indicated. All cultures were incubated without changing the medium for 48 h. Upon harvesting the cultures, levels of total Abeta (panel E) or Abeta (panel F) in the conditioned medium were determined by ELISA. Heparin-Sepharose pellets containing the total s-APP precleared from the samples of medium prior to ELISA were eluted with SDS sample buffer, and levels of extracellular s-APPalpha (panel A) and s-APPbeta (panel B) were determined by Western blot assay using IgG (see ``Experimental Procedures''). Intracellular s-APPbeta (referred to as c-APPbeta) was also measured in lysates of the cell monolayers (panel D). The results of the foregoing assays were normalized to total intracellular SWbetaAPP. In panel C, the values for s-APPalpha and s-APPbeta in the medium are also expressed as a direct ratio. All values are means (± S.E.) of separate determinations performed on three parallel cultures.



In accord with the earlier studies of wild-type betaAPP (Fig. 1B), coexpression of SWbetaAPP with Rab6 resulted in a 35-40% decrease in the amount of Abeta that accumulated in the conditioned medium after 48 h (Fig. 4E). This decrease was similar in magnitude to the decline in s-APPbeta in the same cultures (Fig. 4B). It is now recognized that Abeta exists in a number of isoforms that include alternate carboxyl termini: e.g. Abeta and Abeta(55, 56) . Although Abeta is the more abundant species, Abeta has attracted particular attention since it is prone to form insoluble amyloid fibrils (57) and appears to be a major constituent in amyloid deposits in Alzheimer's disease(58, 59) . Thus, in addition to assaying total Abeta (mostly Abeta), we employed an ELISA that specifically measures Abeta in medium from the cultures expressing SWbetaAPP (Fig. 4F). In contrast to the modest but significant decline in total Abeta observed in cells expressing Rab6versus Rab6 (Fig. 4E), there was no detectable difference in the extracellular Abeta in the same cultures (Fig. 4F).

Fig. 5depicts the results of a separate time-course study in which we confirmed that the increased extracellular s-APPalpha/s-APPbeta ratio in medium from cultures coexpressing Rab6 and SWbetaAPP arises as a result of differential effects of the Rab6 mutant on the rates of secretion of the alpha and beta forms of s-APP (Fig. 5, A-C). The marked increase in the rate of sAPPalpha secretion in the cultures expressing Rab6versus Rab6 was reflected in a 3-fold elevation in the total extracellular s-APPalpha, normalized to intracellular betaAPP, at the 12-h end point (Fig. 5D). In contrast, the cultures expressing Rab6 showed a slight decrease in the normalized value for s-APPbeta (Fig. 5E) and no significant change in Abeta (Fig. 5F).


Figure 5: Differential changes in rates of secretion of s-APPalpha versus s-APPbeta in 293 cells coexpressing SWbetaAPP with Rab6versus Rab6. Beginning 18 h after transfection, fresh medium (2 ml) was added to each culture and aliquots of medium were removed at the designated intervals for quantitation of s-APPalpha (panel A) and s-APPbeta (panel B) by Western blot assay using I-IgG. The direct ratios of the alpha and beta forms of s-APP measured in the medium at 12 h are depicted in panel C. Values for total extracellular s-APPalpha, s-APPbeta (phosphorimager units) and Abeta (picograms) at the 12-h end point were normalized to the total intracellular SWbetaAPP (phosphorimager units) and are depicted in panels D-F. Each value is a mean (± S.E.) of separate determinations performed on three parallel cultures. As in the case of wild-type betaAPP (Fig. 3), immunoblot assays performed on the cell monolayers harvested at the 12-h end point indicated that the intracellular levels of mature and immature SWbetaAPP were comparable in the cultures expressing Rab6 and Rab6 (not shown).




DISCUSSION

The results described in this report demonstrate that overexpression of a GTP-binding-defective Rab6 mutant in 293 cells results in a marked enhancement of secretion s-APPalpha, while extracellular accumulation of products derived from the amyloidogenic processing pathway (i.e. s-APPbeta and Abeta) is either modestly inhibited or unaffected. Extensive studies have documented the localization of Rab6 in the trans-Golgi cisternae and trans-Golgi network in mammalian cells(30, 31, 32) . Therefore, our findings indicate that intra-Golgi transport events regulated by Rab6 selectively influence the routing of betaAPP into the alpha-secretase pathway. While previous studies have shown that s-APPalpha and s-APPbeta are sorted differently in polarized MDCK cells (52) and that secretion of s-APPalpha and Abeta respond in opposite ways to stimulation of protein kinase C(60, 61, 62, 63, 64) , the present report provides the first genetic evidence implicating a specific protein (Rab6) and a specific subcellular compartment (the trans-Golgi network) in the branching of the alpha and beta-secretase pathways for betaAPP processing.

Our studies with Rab8 underscore the specificity of the results obtained with the Rab6 mutant. In contrast to the stimulatory effect of Rab6 on s-APPalpha secretion, Rab8 had a small inhibitory effect on the same pathway. The decreased secretion of s-APPalpha caused by the Rab8 mutant in 293 cells is consistent with the proposed role of Rab8 in transport of proteins between the Golgi apparatus and the basolateral plasma membrane in polarized epithelial cells(50) . However, because the effects of Rab8 were relatively minor in comparison to the complete block in s-APP secretion previously observed when ER Golgi transport was disrupted by Rab1B(27) , we speculate that post-Golgi secretory transport in non-polarized cells probably involves members of the Rab family in addition to Rab8.

In addition to providing mechanistic insights into betaAPP processing, the finding that the dominant-negative N126I mutation in Rab6 stimulates rather than inhibits secretion of s-APPalpha augments the only other mutagenic analysis of Rab6 function performed in intact cells. Specifically, Martinez et al.(33) showed that overexpression of an activating Rab6 mutant, Rab6, caused a delay in constitutive protein secretion, with a corresponding accumulation of transport markers in late Golgi compartments. The latter findings were interpreted as indicating that Rab6 acts either as an inhibitor of anterograde transport through the trans-Golgi compartment or as a positive regulator of retrograde transport from post-Golgi vesicles back to the trans-Golgi network or Golgi cisternae. Our results support this model of Rab6 function, since it would predict that a dominant-negative Rab6 mutant such as Rab6 might facilitate the anterograde flow of glycoproteins into the distal portion of the constitutive secretory pathway where alpha-secretase is thought to reside.

Based on the foregoing model of Rab6 function in intra-Golgi trafficking, we propose that by impairing retrograde transport or facilitating anterograde transport within the trans-Golgi compartment, Rab6 increases the flow of betaAPP into the constitutive alpha-secretory pathway. The absence of a parallel stimulatory effect on secretion of s-APPbeta and Abeta suggests that the pool of betaAPP destined for processing by beta-secretase is sorted into a distinct trans-Golgi or endosomal compartment prior to the transport steps mediated by Rab6. This proposal is consistent with studies indicating that beta-secretase operates in an acidic compartment (16, 17, 18, 19, 65) . An alternative model of amyloidogenic processing envisions that betaAPP is cleaved by beta-secretase and -secretase in endosomes or lysosomes after it escapes cleavage by alpha-secretase and is reinternalized from the plasma membrane. Support for this model is derived from studies showing that intact betaAPP exists at the cell surface and is reinternalized in clathrin-coated endocytic vesicles (12, 65) . However, it seems unlikely that this is the major pathway for biogenesis of Abeta in 293 cells, since we failed to observe a coordinate increase in s-APPbeta and Abeta production in cells where transport of betaAPP to the cell surface was enhanced 2.5-3-fold.

The general experimental approach described herein might be particularly useful in future studies aimed at elucidating the molecular mechanisms underlying the biogenesis of distinct forms of Abeta. Specifically, the mutagenesis of various Rab proteins localized in different segments of the exocytic and endocytic pathways might provide insight into the question of whether Abeta and Abeta arise through the action of unique -secretase activities residing in separate subcellular compartments. While the differential effect of Rab6 on Abetaversus Abeta observed in the present study (Fig. 4) could be consistent with the latter possibility, the results are inconclusive because the effects of Rab6 on total Abeta were small and somewhat variable (e.g. no decrease was observed in the short term studies depicted in Fig. 3and Fig. 5). Thus, it will be of considerable interest to see whether more definitive differences in production of the Abeta isoforms will be detected as these studies are expanded to include additional members of the Rab protein family.


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.

§
These authors contributed equally to the work

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; wam{at}smtp.geisinger.eduger.edu.

(^1)
The abbreviations used are: Abeta, amyloid beta-peptide; beta-APP, beta-amyloid precursor protein; s-APP, soluble amino-terminal exodomain derived from betaAPP; ER, endoplasmic reticulum; PCR, polymerase chain reaction; HEK, human embryonal kidney cells; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; WT, wild-type.

(^2)
F. Castellano and W. A. Maltese, unpublished observation.


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