The role of the Ras-related GTP-binding protein, Rab1B, in intracellular trafficking of beta-amyloid precursor protein (beta APP) was studied in cultured 293 cells. beta APP is processed via one of two alternative routes. In the major secretory pathway, beta APP is cleaved by alpha-secretase within the region comprising the beta-amyloid peptide (A beta), resulting in release of a soluble NH2-terminal exodomain (APP alpha) and a 3-kDa peptide (p3) derived from the carboxyl-terminal tail. In the alternative amyloidogenic pathway, beta APP is cleaved by beta-secretase, with the release of a truncated exodomain (APP beta) and an intact A beta peptide. When beta APP751 was coexpressed with Rab1B(wt) or dominant-negative Rab1B mutants (Rab1BN121I or Rab1BS22N) there was a marked decrease in conversion of the immature Endo-H sensitive form of beta APP751 (108 kDa) to the mature O-glycosylated form of beta APP751 (130 kDa) in cells expressing the mutant forms of Rab1B. The block in Golgi-dependent processing of beta APP was accompanied by inhibition of secretion of APPS (APP alpha). A similar decrease in secretion of APPS (APP alpha+APP beta) was observed in cells that were coexpressing Rab1BN121I with the "Swedish" variant of beta APP751 (i.e. beta APPSW751), which undergoes increased amyloidogenic processing. Coincident with the decline in APPS secretion, the cells coexpressing beta APPSW751 with Rab1BN121I showed a 90% decrease in A beta secretion. The data indicate that Rab1B plays a key role in endoplasmic reticulum-->Golgi transport of beta APP, and that beta APP must pass through a late Golgi compartment before entering either the alpha-secretase or the amyloidogenic beta-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 beta APP transport involved in the biogenesis of A beta.
The Ras-related GTP-binding protein, Rab6, is localized in late Golgi compartments where it mediates intraGolgi vesicular trafficking. Herein we report that coexpression of Alzheimer's -amyloid precursor protein (APP 751 ) with a dominant-negative Rab6 mutant (Rab6 The 4-kDa amyloid -peptide (A) 1 has been implicated in the pathogenesis of Alzheimer's disease (1, 2). A is formed as the result of intracellular proteolytic processing of -amyloid precursor proteins (APP 695 , APP 751 , and APP 770 ). The biogenesis of A begins when APP is cleaved by an enzymatic activity termed -secretase, releasing a soluble NH 2 -terminal exodomain (s-APP), which is secreted from the cell, and leaving a membrane-anchored COOH-terminal fragment containing the intact A sequence (3-6). A is released when the latter fragment is further trimmed by another proteolytic activity currently referred to as ␥-secretase (7, 8). While amyloidogenic processing of APP is known to occur at low levels ubiquitously, cells that produce APP 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 APP within the A domain by an enzymatic activity termed ␣-secretase, releasing a soluble exodomain (s-APP␣) containing part of the A sequence (3, 9 -11). Because the residual COOH-terminal stump lacks an intact A domain, it cannot give rise to A when cleaved by ␥-secretase.The mechanisms by which cells control the flux of APP 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 ␣-secretase acts on the mature form of APP 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 APP by -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 A (16 -19). In particular, a recent study suggests that -secretase cleavage of APP 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 APP, 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 (21-23). Like other Ras-related proteins, R...
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