Mapping the Structural Topology of the Yeast 19S Proteasomal Regulatory Particle Using Chemical Cross-linking and Probabilistic Modeling

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1–6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.Basic cellular homeostasis depends on the regulated protein degradation and turnover by the ubiquitin-proteasome system (1, 2). Central to this pathway is the 26S proteasome complex, which is responsible for ubiquitin/ATP-dependent protein degradation (3–5). The 26S holocomplex is a megadalton-sized protein assembly consisting of the 20S catalytic core particle (CP)¹ and the 19S regulatory particle (RP). The eukaryotic 20S CP is composed of two copies of 14 nonidentical subunits (α_1–7 and β_1–7) arranged into four stacked heptameric rings in an order of α₇β₇β₇α₇. The crystal structure and topology of the highly ordered 20S CP has been resolved and is evolutionarily conserved (6). Although α subunits of the 20S CP are essential for the assembly of the complex and its interactions with the regulatory complex, three catalytic β subunits (β1, β2, and β5) harbor various catalytic activities responsible for regulated proteasomal degradation. The 19S RP is composed of 19 subunits, which forms two subcomplexes, the base consisting of six related AAA-ATPase (Rpt1–6) and four non-ATPase (Rpn1, Rpn2, Rpn10, and Rpn13) subunits and the lid containing nine non-ATPase subunits (Rpn3, Rpn5–9, Rpn11, Rpn12, and Rpn15/Sem1) (7, 8). In comparison with the 20S core, the function and structure of the 19S RP is much less well understood. Nevertheless, it is believed that the 19S RP is involved in multiple functions including recognition of polyubiquitinated substrates (9, 10), cleavage of the polyubiquitin chains to recycle ubiquitin (11), unfolding of substrates, assisting in opening the gate of the 20S chamber, and subsequently translocating the unfolded substrates into the catalytic chamber (4, 12–14). The six AAA-ATPase subunits (Rpt1–6), which directly interact with the 20S α-ring, function as a molecular chaperone responsible for protein unfolding and are involved in substrate translocation and modulating gating of the CP (5, 15). Although detailed functions for most of the 19S non-ATPase subunits remain elusive, Rpn11 is known to carry an Mpr1p and Pad1p N-termini (MPN) domain, which harbors an essential deubiquitination activity responsible for cleaving polyubiquitin chains from proteasomal substrates (11, 16). In addition, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors, which are important in docking ubiquitinated substrates to the proteasome for degradation (4). Moreover, the two largest proteasome subunits, Rpn1 and Rpn2, interact with a variety of proteins including ubiquitin receptors and deubiquitinases and thus may function as scaffolding proteins to assist proteasomal degradation. Thus far, no atomic resolution structures are available for either the 19S RP or the 26S holocomplex. New insights of the overall topology of the 19S RP will illuminate protein interactions within, thus providing evidence for its otherwise unknown functions.Although many studies have been performed to characterize the 19S structure utilizing various techniques including cryo-EM (17, 18) and native mass spectrometry (19), details on spatial interfaces and subunit interconnectivity of the 19S RP remain to be unraveled. During the course of our study, the rough topology of the 19S RP was determined by cryo-EM alone (20) or coupled with other approaches (21); nevertheless more detailed information at the peptide or atomic level is still required. In addition to technological limitations in current approaches, the highly dynamic and heterogeneous nature of the 19S RP may attribute to the difficulty in obtaining its high resolution structure. In recent years, chemical cross-linking coupled with mass spectrometry (XL-MS) has become an attractive alternative for structure analysis of proteins and protein complexes (22, 23). The ability of XL-MS to identify interaction interfaces between proteins allows us to define low resolution protein topology. In addition to protein interaction networks and the site of protein interactions at binding interfaces, cross-linking analysis can reveal information about the spatial distance between cross-linked amino acids on the surface of folded proteins. Although such knowledge only reveals the maximum distance given by the length of the cross-linker and can be influenced by protein conformational flexibility, it can be used as the distance constraint for molecular modeling of protein folds and complex topologies, i.e., the arrangement of the constituents of a complex in space. A recent study by Chen et al. (24) on yeast RNA polymerase II (RNAPII) complex has exemplified the power of XL-MS in elucidating the architecture of large multisubunit complexes. Although effective, cross-linking studies have been challenging because of the low abundance of cross-linked products and the inherent complexity of sequencing interlinked peptides by MS for unambiguous identification. To facilitate MS detection and identification of cross-linked products, we have recently developed a novel homobifunctional amine reactive, low energy MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO), and successfully applied it to cross-link the yeast 20S proteasome for rapid, accurate, and simplified determination of protein interaction interfaces within the complex (25). The unique functionality of our cross-linking reagent and specialized bioinformatics tools significantly increase our confidence and speed in the identification of cross-linked products when compared with cross-linking studies using traditional noncleavable reagents. Current cross-linking studies have been focused on protein complexes with known crystal structures, but topological structures of protein complexes based primarily on cross-linking data have not yet been reported. This is due to the lack of computational tools that use cross-linking data to deduce the spatial organization of subunits in a given complex. To define the architecture of the yeast 19S RP, we have characterized the proximity and interconnectivity of the subunits by employing our newly developed cross-linking strategy. The resulting cross-linking information serves as a basis for a rigorous probabilistic analysis to obtain the maximum likelihood (ML) topology. This strategy is developed by first analyzing our cross-linking data for the 19S six-member AAA-ATPase base ring, as the topology ordering of yeast orthologs has been recently determined (14, 26–28). The effectiveness of this new probabilistic platform is supported by the agreement between our derived ML topology of the AAA-ATPase base ring and previous reports. When the same probabilistic approach is applied to the 19S lid subcomplex, the resulting topology is also in agreement with recently proposed models (20, 21). This work represents the first application of probabilistic modeling of protein complexes based solely on cross-link data, establishing a new workflow for future structural analysis of large protein complexes using XL-MS.     

Subcomplexes of PA700, the 19 S Regulator of the 26 S Proteasome, Reveal Relative Roles of AAA Subunits in 26 S Proteasome Assembly and Activation and ATPase Activity

We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1–6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.The 26 S proteasome is a 2,500,000-Da protease complex that degrades polyubiquitylated proteins by an ATP-dependent mechanism (1, 2). The biochemical processes required for this function are divided between two subcomplexes that compose the holoenzyme (3, 4). The first, called 20 S proteasome or core particle, is a 700,000-Da complex that catalyzes peptide bond hydrolysis (5). The second, called PA700 or 19 S regulatory particle, is a 700,000-Da complex that mediates multiple aspects of proteasome function related to initial binding and subsequent delivery of substrates to the catalytic sites of the 20 S proteasome (6). The 20 S proteasome is composed of 28 subunits representing the products of 14 genes arranged in four axially stacked heteroheptameric rings (7, 8). Each of the two center β rings contains three different protease subunits that utilize N-terminal threonine residues as catalytic nucleophiles (5, 8, 9). These residues line an interior lumen formed by the stacked rings and thus are sequestered from interaction with substrates by a shell of 20 S proteasome subunits.PA700 is composed of 20 different subunits. Six of these subunits, termed Rpt1–6, are AAA² (ATPases Associated with various cellular Activities) family members that confer ATPase activity to the complex and mediate energy-dependent proteolysis by the 26 S proteasome (2, 10). 26 S proteasome assembly from PA700 and 20 S proteasome requires ATP binding to Rpt subunits (11–15). Binding of PA700 to the 20 S proteasome occurs at an axial interface between a heterohexameric ring of the PA700 Rpt subunits and the heteroheptameric outer ring of α-type 20 S proteasome subunits (16). Substrates enter the proteasome through a pore in the center of the α subunit ring that is reversibly gated by conformationally variable N-terminal residues of certain α subunits in response to PA700 binding (12, 17–19). Although the degradation of polyubiquitylated proteins requires additional ATP hydrolysis-dependent actions by PA700, the assembled 26 S proteasome displays greatly increased rates of energy-independent degradation of short peptides by virtue of their increased access to catalytic sites via diffusion through the open pore (15, 18, 20).Recently, specific interactions between Rpt and α subunits that determine PA700-20 S proteasome binding and gate opening have been defined. These findings established nonequivalent roles among the six different Rpt subunits for these processes (12, 19). For example, carboxypeptidase A treatment of PA700 selectively cleaves the C termini of two Rpt subunits (Rpt2 and Rpt5) and renders PA700 incompetent for proteasome binding and activation (19). Remarkably, short peptides corresponding to the C terminus of either Rpt2 or Rpt5, but none of the other Rpt subunits, were sufficient to bind to the 20 S proteasome and activate peptide substrate hydrolysis by inducing gate opening (12, 15, 18). The C-terminal peptides of Rpt2 and Rpt5 appear to bind to different and distinct sites on the proteasome and produce additive effects on rates of peptide substrate hydrolysis, suggesting that pore size or another feature of gating can be variably modulated (19). These various results, however, do not specify whether the action of one or the other or both C-terminal peptides is essential for function of intact PA700.In addition to its role in activation, PA700 plays other essential roles in 26 S proteasome function related to substrate selection and processing. For example, PA700 captures polyubiquitylated proteins via multiple subunits that bind polyubiquitin chains (21–23). Moreover, to ensure translocation of the bound ubiquitylated protein through the narrow opened substrate access pore for proteolysis, PA700 destabilizes the tertiary structure of the protein via chaperone-like activity and removes polyubiquitin chains via deubiquitylating activities of several different subunits (24–30). These various functions appear to be highly coordinated and may be mechanistically linked to one another and to the hydrolysis of ATP by Rpt subunits during substrate processing.Despite support for this general model of PA700 action, there is a lack of detailed knowledge about how PA700 subunits are structurally organized and functionally linked. Previously, we identified and characterized a subcomplex of PA700 called “modulator” that contained two ATPase subunits, Rpt4 and Rpt5, and one non-ATPase subunit, p27 (31). Although this protein was identified by an assay that measured increased PA700-dependent proteasome activation, the mechanistic basis of this effect was not clear. Moreover, the modulator lacked detectable ATPase activity and proteasome activating activity. The latter feature is surprising in retrospect because of the newly identified capacity of Rpt5 to activate the proteasome directly (12, 19). This disparity suggests that specific interactions among multiple PA700 subunits determine the manifestation and regulation of various activities.This study extends our recent findings regarding relative roles of Rpt subunits in the regulation of proteasome function. It also provides new insights and significance to older work that identified and characterized the modulator as a subcomplex of PA700. Our findings unite two different lines of investigation to offer new information about the structure, function, and regulation of 26 S proteasome. They also offer insights about alternative models for assembly of PA700 and 26 S proteasome in intact cells.     

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets.Although first described over a century ago, new roles and functions for platelets continue to emerge. Derived by budding from megakaryocytes and devoid of a nucleus, platelets were formerly not thought to produce proteins and their one role was to initiate and perform blood clotting. However, this view has changed in recent years; platelets have mRNA, microRNAs to regulate their mRNA, the machinery to synthesize proteins and they use it (1, 2). Furthermore, in addition to their function in hemostasis, it has been recognized that platelets play a role in inflammatory processes (3, 4). Through their interactions with the endothelium and other blood cells, platelets are believed to play a critical role in defense, wound repair, and more (5). Understanding of many of the new aspects of platelet function is still limited, but these recent advances raise the question of what other features are awaiting discovery that might be hidden in these small cell fragments.There are limited methods available with which to study platelets; DNA-based methods cannot be applied, and although mRNA is present in platelets, its low level only allows for restricted analysis. Mass spectrometry (MS)-based proteomics is particularly well set up to study platelets, and previous studies have analyzed the platelet proteome (6–11), various subproteomes (12–16), and have shed light on aspects of platelet signaling and function (17–21). In this study, proteomic analysis of human platelets was conducted, generating an inventory of platelet proteins, which was then explored by comparison to proteomic data sets of nucleated cells with the aim of identifying new biology-related functions. This approach revealed consistently high expression of the proteasome, the protein complex that is the main protein degradation machinery in cells (Fig. 1). The presence of the proteasome in platelets has been described earlier (22). It is known to be active and its activity increases in response to agonist stimulation (23); however, a detailed analysis of the many subunits of this multimeric complex has not been performed and its role in platelets, which produce less protein than nucleated cells, is not fully understood. The proteasome''s core complex, the 20S proteasome, is composed of 28 nonidentical subunits, arranged in four rings, two comprising of seven α subunits and two of seven β subunits. Three of the β subunits (β1, β2, and β5) are catalytically active. The 20S proteasome forms the 26S proteasome together with the 19S regulator, which contains ATPase subunits and is responsible for the ATP¹ dependence of the 26S proteasome. The immunoproteasome, which is constitutively expressed in cells of the immune system or is synthesized following induction by interferon γ (IFNγ) in all other nucleated cells, is formed when the catalytically active β subunits are replaced by their immunoproteasome counterparts (β1i, β2i, and β5i). IFNγ also up-regulates the 11S regulator, which consists of PA28 α and β subunits, and both the immunoproteasome and the 11S proteasome are thought to be involved in improved peptide generation for major histocompatibility complex (MHC) I antigen presentation (24).Open in a separate windowFig. 1.Composition of the proteasome and immunoproteasome. The standard 20S core (middle) is composed of 28 nonidentical subunits that are arranged in four rings; two composed of seven α subunits and two composed of seven β subunits. Three of the β subunits (β1, β2, and β5) are catalytically active. The 19S regulator is composed of a base, containing six ATPase subunits and two non-ATPase subunits, and a lid, which contains up to ten non-ATPase subunits. The 20S proteasome and two 19S regulators form the 26S proteasome (left). The immunoproteasome, which is induced by IFNγ, contains three different catalytically active subunits (β1i, β2i, and β5i). The 11S regulator, which consists of heptameric complexes containing PA28α and β subunits, is also induced by IFNγ and can replace the 19S regulator (right).Here, discovery of the high expression of the proteasome in our platelet proteomic data set was followed up with traditional biochemical assays to explore in detail the composition of the proteasome in platelets. Not only were all components of the 26S proteasome detected in our global platelet data sets, but immunoproteasome subunits were also identified. We validated that all members of the 20S proteasome were present and assembled in human platelets. Furthermore, we show that the standard as well as the immunoproteasome catalytic subunits are active. The presence of not only active proteasome but active immunoproteasome subunits in platelets opens up the possibility of new roles for these anucleate players, and further illustrates the critical role proteomics plays in improving our understanding of platelet function.     

Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase

In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ∼2.5-MDa (1) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2, 3). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4, 5). The 19 S regulatory particle consists of at least 17 different subunits (1, 6). Nine of these subunits form a “lid,” whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7–10) as well as cross-linking experiments (11, 12) have demonstrated that the six homologous ATPases are associated with the α rings of the 20 S particle.Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15). In both PAN and the 19 S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the α ring through which substrates enter the 20 S particle (15–17). Because this portal is quite narrow (18–20), only extended polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded by the associated ATPase complex to be translocated and digested within the 20 S particle.PAN and the six ATPases found at the base of the 19 S particle are members of the AAA+ superfamily of multimeric ATPases which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (8, 21). For mechanistic studies of the roles of ATP, the simpler archaeal PAN-20 S system offers many technical advantages over the much more complex 26 S proteasome. For example, prior studies of PAN (17, 22) demonstrated that unfolding of globular substrates (e.g. green fluorescent protein-ssrA) requires ATP hydrolysis. The same was also shown for the Escherichia coli ATP-dependent proteases ClpXP (23) and ClpAP (24). We have also shown that unfolding by PAN can take place on the surface of the ATPase ring in the absence of translocation (15). Thus, unfolding seems to proceed independently from protein translocation into the 20 S proteolytic particle. It is noteworthy that other studies suggest that proteins are unfolded by energy-dependent translocation through the ATPase ring (25, 26). These studies have suggested that the translocation of an unfolded polypeptide from the ATPase into the 20 S core is an active process that is coupled to ATP hydrolysis. A key to underline a detailed molecular mechanism for substrate binding, unfolding, and translocation by the proteasome regulatory ATPase complex is improved understanding of its architecture and the nucleotide-dependent structural transitions that afford these functions.To date we and others have failed to generate micrographs suitable for three-dimensional reconstruction of PAN using single-particle EM analysis. Likewise, structural information regarding the three-dimensional architecture and subunit organization within the 19 S particle is very limited. In fact, high resolution three-dimensional information on the 19 S complex is not yet available. Most knowledge available is based on cross-linking experiments (11, 12) as well as EM structural analysis (7–10), which provided a three-dimensional model outline of the general architecture of the 26 S complex. Unlike the 19 S complex, the structure of the 20 S subcomplex was determined by x-ray crystallography (18, 19). In contrast to the highly homogenous structure of the 20 S complex, the structural heterogeneity and flexibility of the 19 S subcomplex is presumably reflected in multiple conformations, which in turn also contribute to the difficulty in generating a high resolution three-dimensional structural model of the 26 S proteasome. Accordingly, the initial goal of this study was to generate a three-dimensional model of PAN that will allow us to determine its general architecture and to correlate unique conformational transitions within this ATPase with the nucleotide state of the complex (i.e. in the presence of ATPγS, ADP, or in the absence of nucleotides).Smith et al. (27) suggested a general architecture for the PAN-20 S complex based on two-dimensional averaging of a Thermoplasma acidophilum (TA)³ 20 S proteasome and Methanococcus jannaschii (MJ) PAN hybrid complex in the presence of ATPγS. Based on side-view projections of that complex, these authors proposed that PAN assumes an overall structure similar to E. coli HslU (28–30).We realized that although PAN appears heterogeneous in electron micrographs, it does not occupy all possible orientations when adsorbed to carbon-coated electron microscopy (EM) grids, a prerequisite for single particle analysis. This problem was overcome by applying electron tomography in conjunction with a three-dimensional averaging procedure that accounts for the missing wedge in the Fourier space of electron tomograms (31, 32). The three-dimensional model generated revealed an unexpected architecture leading to a possible molecular mechanism describing the function of PAN and presumably the 19 S ATPases.     

APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)⁵ protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants.