γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Summary Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B and mice knocked-out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knock-down of AP-1A causes missorting of basolateral proteins in MDCK cells but only after knock-down of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN and its knock-down promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of TfR and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination, and contains an intrinsic ubiquitin E3 ligase activity, all of which require an N-terminal A20 zinc finger and an immediately C-terminal helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5 Å resolution shows that Rabex-5:ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin binding domain, an inverted ubiquitin interaction motif (IUIM), that binds with ~29 μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ~22 μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc finger diaromatic patch mediates E3 ligase activity by directly recruiting a ubiquitin-loaded ubiquitin conjugating enzyme.The Rab GTPases are central regulators of vesicular trafficking and organelle identity in all eukaryotes 1,2 . The Rab family is the largest branch of the Ras superfamily, comprising more than 60 members in mammalian cells. Like other small GTPases, the localization and activity of the Rab proteins is regulated by GTPase activating proteins (GAPs), guanine nucleotide dissociation inhibitors (GDIs), and guanine nucleotide exchange factors (GEFs) 3,4 . Rab GEFs promote the binding of GTP to Rab proteins, which in turn converts them to their active signaling conformation, and stabilizes their binding to cellular membranes. The founding member of the Rab5 GEF family is the yeast vacuolar sorting protein Vps9 5 . Vps9 is the yeast ortholog of the human Rab5 GEF Rabex-5. All Rab5 GEFs have in common a catalytic unit comprising a helical bundle and a Vps9 homology domain 6 . Most Rab5 GEFs do not function alone, but rather as components of larger multiprotein complexes, as exemplified by the Rabaptin-5-Rabex-5 complex 7-10 .Covalent monoubiquitination of proteins is a major regulatory signal in protein trafficking 11 . In this process, the C-terminal carboxylate of a single molecule of the highly conserved 76-amino acid protein ubiquitin is covalently linked to a Lys residue in a substrate protein.correspondence should be addressed to James H. Hurley at hurley@helix.nih.gov.. 4 Y. C. T. and R. M. contributed equally.Coordinates. The crystallographic coordinates have been deposited in the protein data bank with accession code ____ (to be provided). NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2006 September 26. Published in final edited form as:Nat Struct Mol Biol. 2006 March ; 13(3): 264-271. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis reaction is carried out by a series of enzymes known as E1, E2, and E3 12-14 . Monoubiquitination of many transmembrane cargo proteins marks them for sorting into endosomal pathways 15-17 . Monoubiquitin moieties on these proteins are recognized by specific ubiquitin binding domains in proteins of the tr...

AP-4 is a member of the heterotetrameric adaptor protein (AP) complex family involved in protein sorting in the endomembrane system of eukaryotic cells. Interest in AP-4 has recently risen with the discovery that mutations in any of its four subunits cause a form of hereditary spastic paraplegia (HSP) with intellectual disability. The critical sorting events mediated by AP-4 and the pathogenesis of AP-4 deficiency, however, remain poorly understood. Here we report the identification of ATG9A, the only multispanning membrane component of the core autophagy machinery, as a specific AP-4 cargo. AP-4 promotes signal-mediated export of ATG9A from the -Golgi network to the peripheral cytoplasm, contributing to lipidation of the autophagy protein LC3B and maturation of preautophagosomal structures. These findings implicate AP-4 as a regulator of autophagy and altered autophagy as a possible defect in AP-4-deficient HSP.