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.

Sorting of transmembrane proteins to endosomes, lysosomes, lysosome-related organelles, and the basolateral plasma membrane of polarized epithelial cells is driven by the recognition of signals in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats (1-4). Key components of this system are the heterotetrameric adaptor protein (AP) 4 complexes, AP-1 (␥-␤1-1-1), AP-2 (␣-␤2-2-2), AP-3 (␦-␤3-3-3), and AP-4 (⑀-␤4-4-4) (subunit composition shown in parentheses) (see Fig. 1) (1-4). AP-1, AP-2, and AP-3 associate with clathrin, whereas AP-4 is most likely part of a nonclathrin coat. Another property common to AP-1, AP-2, and AP-3, but not AP-4 is their potential heterogeneity due to the existence of multiple subunit isoforms encoded by different genes, including two ␥ (␥1 and ␥2), two 1 (1A and 1B), and three 1 (1A, 1B and 1C) for AP-1; two ␣ (␣A and ␣C) for AP-2; and two ␤3 (␤3A and ␤3B), two 3 (3A and 3B), and two 3 (3A and 3B) for AP-3 (1). In addition, ␤1 can substitute for ␤2 in the AP-2 complex (5-6), the only known case in which a subunit of one AP complex can be incorporated into another. Thus, combinatorial assembly of different subunit isoforms could give rise to twelve AP-1, four AP-2, and eight AP-3 complexes (see Fig. 1). It is not known, however, whether most of these combinations occur in cells and whether particular subunit isoforms endow the complexes with different functional properties.AP-1, AP-2, and AP-3 recognize sorting signals fitting the "tyrosine-based," YXXØ, and "dileucine-based," (D/E)XXXL(L/I) consensus motifs (where Ø is an amino acid with a bulky hydrophobic side chain, i.e. leucine, isoleucine, methionine, valine, or phenylalanine) (2-4, 7). Although both types of signals play similar roles in protein sorting, they bind to different sites on the AP complexes. Yeast two-hybrid and other protein interaction assays showed that YXXØ signals bind to the subunits of AP-1, . (D/E)XXXL(L/I) signals, on the other hand, do not bind to any single AP subunit but to combinations of ␥-1, ␣-2, and ␦-3 subunits, as demonstrated by the use of yeast three-hybrid (Y3H) and in vitro binding assays (15-17).X-ray crystallographic analyses have shed light on the structural basis for the interactions of YXXØ and (D/ E)XXXL(L/I) signals with the AP-2 complex (18 -20). Both binding sites are located on the AP-2 "core," a domain formed by the amino-terminal regions of ␣ and ␤2, and the entire 2 and 2 subunits (Fig. 1). The YXXØ-binding site comprises two hydrophobic pockets on 2 that accommodate the Y and Ø residues of the signals (18). The binding site for (D/ E)XXXL(L/I) signals likely corresponds to that of a dileucinecontaining "Q-peptide" from CD4, (RMpSQIKRLLSE), which was recently identified by Kelly et al. (20). The Q-peptide does not strictly conform to the definition of a (D/E)XXXL(L/I) signal, although the Gln residue at position Ϫ4 or the phos-

Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still unde®ned set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin5±Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439±443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the g1-and g2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA±Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green¯uorescent protein (GFP)±Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gadaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

SUMMARY Plasma membranes of the somatodendritic and axonal domains of neurons are known to have different protein compositions, but the molecular mechanisms that determine this polarized protein distribution remain poorly understood. Herein we show that somatodendritic sorting of various transmembrane receptors in rat hippocampal neurons is mediated by recognition of signals within the cytosolic domains of the proteins by the µ1A subunit of the adaptor protein-1 (AP-1) complex. This complex, in conjunction with clathrin, functions in the neuronal soma to exclude somatodendritic proteins from axonal transport carriers. Perturbation of this process affects dendritic spine morphology and decreases the number of synapses. These findings highlight the primary recognition event that underlies somatodendritic sorting and contribute to the evolving view of AP-1 as a global regulator of cell polarity.

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as “AP-4 deficiency syndrome”. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.