Mutations in presenilins 1 and 2 (PS1 and PS2) account for the majority of cases of early-onset familial Alzheimer's disease. However, the trafficking and interaction of PSs with other proteins in the early secretory pathways are poorly understood. Using co-immunoprecipitation, we found that PS bound to Syx5 (syntaxin 5), which is a target-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor involved in endoplasmic reticulum (ER)-Golgi vesicular transport in vivo. Syx5 interacted only with the full-length PS holoproteins and not with the naturally occurring N- or C-terminal fragments. The PS holoproteins co-immunoprecipitated with the mutant Syx5, which localized to the ER and Golgi compartments, despite the substitution of the transmembrane region with that of syntaxin 1A. In contrast, the transmembrane deletion mutant that localized to the cytosol, but not to the ER or Golgi compartments, did not co-immunoprecipitate the PS holoproteins. The PS1 variant linked to familial Alzheimer's disease (PS1DeltaE9), lacking the region that contains the endoproteolytic cleavage site in the cytoplasmic loop, showed markedly decreased binding to Syx5. Immunofluorescence and sucrose-density-gradient fractionation analyses showed that the full-length PS holoproteins co-localized with Syx5 to the ER and cis-Golgi compartments. Furthermore, Syx5 overexpression resulted in the accumulation of PS holoproteins and the beta-amyloid precursor protein, and reduced the secretion of the Abeta (amyloid beta) peptide in COS-7 cells. In summary, these results indicate that Syx5 binds to full-length PSs and affects the processing and trafficking of beta-amyloid precursor protein in the early secretory compartments.

Rab family small GTP ases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP ‐bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG 6 ( BAT 3/Scythe) predominantly recognizes a cryptic portion of GDP ‐associated Rab8a, while its major GTP ‐bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG 6 and the degradation of GDP ‐Rab8a via the ubiquitin‐proteasome system. BAG 6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG 6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG 6, and the BAG 6‐mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.

It has been suggested that syntaxin 5 (Syx5) participates in vesicular transport. We examined the effects of Syx5 down-regulation on the morphology of the Golgi apparatus and the transport of vesicles in mammalian cells. Knockdown of the Syx5 gene resulted in Golgi fragmentation without changing the level of endoplasmic reticulum (ER)-resident proteins, other Golgi-SNAREs (soluble N-ethylmaleimide-sensitive factorattachment protein receptors), and coatmer proteins. Strikingly, a major decrease in Syx5 expression barely affected the anterograde transport of vesicular stomatitis virus G (VSVG) protein to the plasma membrane. These results suggest that Syx5 is required for the maintenance of the Golgi structures but may not play a major role in the transport of vesicles carrying VSVG between the ER and the Golgi compartment.

The specific roles of syntaxin 5 (Syx 5) in the interaction with presenilin (PS) and the accumulation of b-amyloid precursor protein (bAPP), as well as the secretion of b-amyloid peptide (Ab peptide) were examined in NG108-15 cells. Syx 5, which localizes from the endoplasmic reticulum (ER) to the Golgi, bound to PS holoproteins, while the other Syxs studied did not. Among familial Alzheimer's disease (FAD)-linked PS mutants, PS1DE9, which lacks the endoproteolytic cleavage site, showed markedly decreased binding to Syx 5. The interaction domains in Syx 5 were mapped to the transmembrane region and to the cytoplasmic region containing the a-helical domains, which are distinct from the H3 (SNARE motif). Among all of the Syxs examined, only overexpression of Syx 5 resulted in the accumulation of bAPP in the ER to cis-Golgi compartment, an attenuation of the amount of the C-terminal fragment (APP-CTF) of bAPP, and a reduction in the secretion of Ab peptides. Furthermore, co-expression of Syx 5 with C99 resulted in an increase in APP-CTF and suppressed Ab secretion. Taken together, these results indicate that Syx 5 may play a specific role in the modulation of processing and/or trafficking of FAD-related proteins in neuronal cells by interaction with PS holoproteins in the early secretory compartment of neuronal cells.

In this study, we examined the interaction of Syntaxin 5L (Syx5L), a Syx5 isoform that has an N-terminal extension containing a di-arginine ER-retrieval motif, with presenilin (PS) and its effects on the processing of beta-amyloid precursor protein (betaAPP). Similar to Syx5, Syx5L bound to PS1 holoprotein but not to its N- or C-terminal fragments. Unlike Syx5, Syx5L overexpression did not cause marked accumulation of intracellular betaAPP holoprotein, and did not inhibit amyloid beta peptide (Abeta) secretion. Analyses using deletion mutants of Syx5L revealed that, in addition to the difference in the intracellular localization between the isoforms, the presence of the N-terminal extension in Syx5L was critical for suppressing its inhibition of betaAPP processing. Treatment of cells that overexpressed Syx5L with brefeldin A, an inhibitor of transport from the ER to the Golgi compartments, resulted in substantial accumulation of intracellular betaAPP holoprotein and reduction in the secretion of Abeta. Although Syx5 and Syx5L share lengthy regions of amino acid identity, they appear to play distinct roles in modulating the metabolism and trafficking of betaAPP in the early secretory compartment.