Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.     

GGA proteins mediate the recycling pathway of memapsin 2 (BACE)

Memapsin 2 (BACE, beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta (A beta) and the progression of Alzheimer disease. Both memapsin 2 and APP are transported from the cell surface to endosomes where APP is cleaved by memapsin 2. We described previously that the cytosolic domain of memapsin 2 contains an acid cluster-dileucine motif (ACDL) that binds the VHS (Vps-27, Hrs, and STAM) domain of Golgi-localized gamma-ear-containing ARF-binding (GGA) proteins (He, X., Zhu, G., Koelsch, G., Rodgers, K. K., Zhang, X. C., and Tang, J. (2003) Biochemistry 42, 12174-12180). Here we report that GGA proteins colocalize in the trans-Golgi network and endosomes with memapsin 2 and a memapsin 2 chimera containing a cytosolic domain of a mannose-6-phosphate receptor. Depleting cellular GGA proteins with RNA interference or mutation of serine 498 to stop the phosphorylation of ACDL resulted in the accumulation of memapsin 2 in early endosomes. A similar change of memapsin 2 localization also was observed when a retromer subunit, VPS26, was depleted. These observations suggest that GGA proteins function with the phosphorylated ACDL in the memapsin 2-recycling pathway from endosomes to trans-Golgi on the way back to the cell surface.     

Specificity of memapsin 1 and its implications on the design of memapsin 2 (beta-secretase) inhibitor selectivity

Memapsin 1 is closely homologous to memapsin 2 (BACE), or beta-secretase, whose action on beta-amyloid precursor protein (APP) leads to the production of beta-amyloid (A beta) peptide and the progression of Alzheimer's disease. Memapsin 2 is a current target for the development of inhibitor drugs to treat Alzheimer's disease. Although memapsin 1 hydrolyzes the beta-secretase site of APP, it is not significantly present in the brain, and no direct evidence links it to Alzheimer's disease. We report here the residue specificity of eight memapsin 1 subsites. In substrate positions P(4), P(3), P(2), P(1), P(1)', P(2)', P(3)', and P(4)', the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe, and Trp, respectively, while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp, and Phe, respectively. Other less preferred residues can also be accommodated in these subsites of memapsin 1. Despite the broad specificity, these residue preferences are strikingly similar to those of human memapsin 2 [Turner et al. (2001) Biochemistry 40, 10001-10006] and thus pose a serious problem to the design of differentially selective inhibitors capable of inhibiting memapsin 2. This difficulty was confirmed by the finding that several potent memapsin 2 inhibitors effectively inhibited memapsin 1 as well. Several possible approaches to overcome this problem are discussed.     

Structural Features of Human Memapsin 2 （β-Secretase） and Their Biological and Pathological Implications

Alzheimer’s disease (AD), which is characterized bythe progressive destruction of brain functions in olderpeople, was first recognized in the early 20th century.Since then, modern medicine has further increased thenumber of people living to old age. AD h…     

Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

Structural locations and functional roles of new subsites S5, S6, and S7 in memapsin 2 (beta-secretase)

Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP), leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease. The active site of memapsin 2 has been shown, with kinetic data and crystal structures, to bind to eight substrate residues (P(4)-P(4)'). We describe here that the addition of three substrate residues from P(7) to P(5) strongly influences the hydrolytic activity by memapsin 2 and these subsites prefer hydrophobic residues, especially tryptophan. A crystal structure of memapsin 2 complexed with a statine-based inhibitor spanning P(10)-P(4)' revealed the binding positions of P(5)-P(7) residues. Kinetic studies revealed that the addition of these substrate residues contributes to the decrease in K(m) and increase in k(cat) values, suggesting that these residues contribute to both substrate recognition and transition-state binding. The crystal structure of a new inhibitor, OM03-4 (K(i) = 0.03 nM), bound to memapsin 2 revealed the interaction of a tryptophan with the S(6) subsite of the protease.     

Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates

Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophe nyl azo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role.     

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].     

Interactions of GGA3 with the ubiquitin sorting machinery

The Golgi-localized, gamma-ear-containing, Arf-binding (GGA) proteins constitute a family of clathrin adaptors that are mainly associated with the trans-Golgi network (TGN) and mediate the sorting of mannose 6-phosphate receptors. This sorting is dependent on the interaction of the VHS domain of the GGAs with acidic-cluster-dileucine signals in the cytosolic tails of the receptors. Here we demonstrate the existence of another population of GGAs that are associated with early endosomes. RNA interference (RNAi) of GGA3 expression results in accumulation of the cation-independent mannose 6-phosphate receptor and internalized epidermal growth factor (EGF) within enlarged early endosomes. This perturbation impairs the degradation of internalized EGF, a process that is normally dependent on the sorting of ubiquitinated EGF receptors (EGFRs) to late endosomes. Protein interaction analyses show that the GGAs bind ubiquitin. The VHS and GAT domains of GGA3 are responsible for this binding, as well as for interactions with TSG101, a component of the ubiquitin-dependent sorting machinery. Thus, GGAs may have additional roles in sorting of ubiquitinated cargo.     

Arf regulates interaction of GGA with mannose-6-phosphate receptor

The role of ADP-ribosylation factor (Arf) in Golgi associated, γ-adaptin homologous, Arf-interacting protein (GGA)-mediated membrane traffic was examined. GGA is a clathrin adaptor protein that binds Arf through its GAT domain and the mannose-6-phosphate receptor through its VHS domain. The GAT and VHS domains interacted such that Arf and mannose-6-phosphate receptor binding to GGA were mutually exclusive. In vivo , GGA bound membranes through either Arf or mannose-6-phosphate receptor. However, mannose-6-phosphate receptor excluded Arf from GGA-containing structures outside of the Golgi. These data are inconsistent with predictions based on the model for Arf's role in COPI veside coat function. We propose that Arf recruits GGA to a membrane and then, different from the current model, 'hands-off' GGA to mannose-6-phosphate receptor. GGA and mannose-6-phosphate receptor are then incorporated into a transport intermediate that excludes Arf .     

Crystal structure of GGA2 VHS domain and its implication in plasticity in the ligand binding pocket

Golgi-localized, gamma-ear-containing, ARF binding (GGA) proteins regulate intracellular vesicle transport by recognizing sorting signals on the cargo surface in the initial step of the budding process. The VHS (VPS27, Hrs, and STAM) domain of GGA binds with the signal peptides. Here, a crystal structure of the VHS domain of GGA2 is reported at 2.2 A resolution, which permits a direct comparison with that of homologous proteins, GGA1 and GGA3. Significant structural difference is present in the loop between helices 6 and 7, which forms part of the ligand binding pocket. Intrinsic fluorescence spectroscopic study indicates that this loop undergoes a conformational change upon ligand binding. Thus, the current structure suggests that a conformational change induced by ligand binding occurs in this part of the ligand pocket.     

Interaction of the cation-dependent mannose 6-phosphate receptor with GGA proteins

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S-transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.     

Enzymic properties of recombinant BACE2.

BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.     

The trihelical bundle subdomain of the GGA proteins interacts with multiple partners through overlapping but distinct sites

The Golgi-localized, gamma-adaptin ear-containing, ARF-binding (GGA) proteins are monomeric clathrin adaptors that mediate the sorting of cargo at the trans-Golgi network and endosomes. The GGAs contain four different domains named Vps27, Hrs, Stam (VHS); GGAs and TOM1 (GAT); hinge; and gamma-adaptin ear (GAE). The VHS domain recognizes transmembrane cargo, whereas the hinge and GAE regions bind clathrin and accessory proteins, respectively. The GAT domain is a polyfunctional module that interacts with various partners including the small GTPase ARF, the endosomal fusion regulator Rabaptin-5, ubiquitin, and the product of the tumor susceptibility gene 101 (TSG101). Previous x-ray crystallographic analyses showed that the GAT region is composed of two subdomains, an N-terminal helix-loop-helix containing the ARF binding site, and a C-terminal triple alpha-helical (trihelical) bundle. In this study, we define the Rabaptin-5 binding site on the GGA1-GAT domain and its relationship to the binding sites for ubiquitin and TSG101. Our observations show that Rabaptin-5, ubiquitin, and TSG101 bind to overlapping but distinct binding sites on the trihelical bundle. The different GAT binding partners engage in both competitive and cooperative interactions that may be important for the function of the GGAs in protein sorting.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1)

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.     

Predicting memapsin 2 (��-secretase) hydrolytic activity

Memapsin 2 (BACE1, β‐secretase), a membrane aspartic protease, functions in the cleavage of brain β‐amyloid precursor protein (APP) leading to the production of β‐amyloid. Because the excess level of β‐amyloid in the brain is a leading factor in Alzheimer's disease (AD), memapsin 2 is a major therapeutic target for inhibitor drugs. The substrate‐binding cleft of memapsin 2 accommodates 12 subsite residues, from P₈ to P₄′. We have determined the hydrolytic preference as relative k_cat/K_M (preference constant) in all 12 subsites and used these data to establish a predictive algorithm for substrate hydrolytic efficiency. Using the sequences from 12 reported memapsin 2 protein substrates, the predicted and experimentally determined preference constants have an excellent correlation coefficient of 0.97. The predictive model indicates that the hydrolytic preference of memapsin 2 is determined mainly by the interaction with six subsites (from P₄ to P₂′), a conclusion supported by the crystal structure B‐factors calculated for the various residues of transition‐state analogs bound to different memapsin 2 subsites. The algorithm also predicted that the replacement of the P₃, P₂, and P₁ subsites of APP from Val, Lys, and Met, respectively, to Ile, Asp, and Phe, respectively, (APP_IDF) would result in a highest hydrolytic rate for β‐amyloid‐generating APP variants. Because more β‐amyloid was produced from cells expressing APP_IDF than those expressing APP with Swedish mutations, this designed APP variant may be useful in new memapsin 2 substrates or transgenic mice for AD studies.     

GGA Autoinhibition Revisited

The cytosolic adaptors GGA1‐3 mediate sorting of transmembrane proteins displaying a C‐terminal acidic dileucine motif (DXXLL) in their cytosolic domain. GGA1 and GGA3 contain similar but intrinsic motifs that are believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues upstream of the DXXLL motif. In the present study, we have subjected the widely acknowledged concept of GGA1 autoinhibition to a thorough structural and functional examination. We find that (i) the intrinsic motif of GGA1 is inactive, (ii) only C‐terminal DXXLL motifs constitute active GGA binding sites, (iii) while aspartates and phosphorylated serines one or two positions upstream of the DXXLL motif increase GGA1 binding, phosphoserines further upstream have little or no influence and (iv) phosphorylation of GGA1 does not affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional significance of GGA autoinhibition in particular and of intrinsic GGA binding motifs in general.     

ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.     

RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of beta-secretase BACE1 in vitro

The beta-amyloid peptide (Abeta) is a major component of the Alzheimer's disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Since BACE1 initiates Abeta generation it represents a valuable target to interfere with Abeta production and treatment of AD. While the enzymatic activity of BACE1 resides in the extracellular domain, the protein also contains a short cytoplasmic tail (B1-CT). This domain serves as a binding site for at least two proteins, the copper chaperone for superoxide dismutase-1 (CCS), and the Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA1) protein, and contains a single phosphorylation site. However, the precise role of the B1-CT for the overall biological function of this protein is largely unknown. Functional studies focusing on the activity of this domain would strongly benefit from the availability of domain-specific inhibitors. Here we describe the isolation and characterization of RNA aptamers that selectively target the B1-CT. We show that these RNAs bind to authentic BACE1 and provide evidence that the binding site is restricted to the membrane-proximal half of the C terminus. Aptamer-binding specifically interferes with the recruitment of CCS, but still permits GGA1 association and casein kinase-dependent phosphorylation, consistent with selective binding site targeting within this short peptide. Because phosphorylation and GGA1 binding to B1-CT regulate BACE1 transport, these RNA inhibitors could be applied to investigate B1-CT activity without affecting the subcellular localization of BACE1.