Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla 1 . Ankyrin repeats contain antiparallel a-helices that can stack to form a superhelical spiral 2 . Visual inspection of the extrapolated structure of 24 ankyrin-R repeats 2 indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles 3-5 . Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common aminoacid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.The atomic structure of 12 ankyrin-R repeats suggests that ankyrin stacks composed of n $ 24 repeats should form a full superhelical turn with putative spring properties 2,3 . We used an atomic force microscope (AFM) to identify individual stacks of 24 ankyrin-B repeats ( Supplementary Fig. S2) and found that they do indeed have a hook-like shape 2 with the molecules' end-to-end distance closely matching the ,12 nm determined for the extrapolated structure 2 (Fig. 1a). Thus, the AFM images strongly suggest that the engineered protein, bearing at its terminus a glutathione S-transferase (GST) module, is correctly folded and does not aggregate. These conclusions are further supported by circular dichroism and hydrodynamic measurements (Supplementary Table 1 and Supplementary Fig. S1).For elasticity measurements, heptahistidine-tagged polypeptides containing 24 ankyrin-B repeats with or without GST, or 12 repeats with GST, were immobilized on a glass surface bearing the metal chelate N-nitrilotriacetic acid (NTA) 6,7 (Fig. 1a). Molecules were stretched vertically, in solution, by the AFM cantilever, and their length and tension were measured with subnanometre and ,10 pN precision [8][9][10] . Most trials revealed complex force-extension profiles with irregularly spaced force peaks typical of multiple molecules ( Supplementary Fig. S4a). However, ,5% of the force-extension curves had simple and consistent features that, we argue, represent LETTERSFigure 1 | Atomic force microscopy measurements reveal the linear elasticity of ankyrin-B repeats. a, The extrapolated structure of 24 ankyrin-R repeats 2 and a diagram of the elasticity measurement on a His-tagged ankyrin fragment bound to NTA (red handles) and stretched with the AFM cantilever. b-e, Force-extension curves of individual ankyrins: 24 repeats with GST (b-d); 24 repeats with no GST (e)....

Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl-␤-cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.Whole body cholesterol homeostasis is maintained through three major pathways: de novo synthesis, intestinal absorption, and biliary excretion. Mice lacking npc1l1 (Niemann-Pick C1-like 1) have a substantial reduction in intestinal cholesterol absorption and are resistant to high cholesterol diet-induced cholesterol accumulation (1-3). The phenotypes of npc1l1-null mice recapitulate the effect of ezetimibe (1, 2), a novel cholesterol absorption inhibitor (4 -6), indicating that NPC1L1 is in the ezetimibe inhibitory pathway. Although both the annexin-2/caveolin-1 complex and aminopeptidase N have been reported previously to be the direct target of ezetimibe (7, 8), caveoilin-1 knockout mice have a normal percentage of cholesterol absorption (9), and the physiological evidence for aminopeptidase N as the ezetimibe target has yet to be shown. On the other hand, ezetimibe was recently shown to specifically bind to NPC1L1 (10). All these data strongly support that NPC1L1 is the target of ezetimibe and resides within the cholesterol uptake pathway. However, the reconstitution of NPC1L1-dependent cholesterol transport in cultured cell systems has been unsuccessful, and tissue-specific cofactors were speculated to be needed (1), limiting further exploration of the molecular basis for cholesterol absorption.The NPC1L1 gene was initially identified to be a homolog of NPC1 (Niemann-Pick C1) and was predicted to be involved in intracellular cholesterol trafficking (11) based on the fact that mutations in the NPC1 gene result in a lipid storage disease, Niemann-Pick disease type C1 (12, 13). NPC1L1 is widely expressed in many human tissues, with the highest expression in the liver and small intestine (1, 3, 11). The expression pattern varies among species. Mouse and rat npc1l1 mRNAs are much more abundant in the small intestine than in the liver (1, 3). The reason for the different tissue expression patterns among species is unknown.T...

Atherosclerosis, which underlies life-threatening cardiovascular disorders including myocardial infarction and stroke 1 , is initiated by low density lipoprotein cholesterol (LDL) passage into the artery wall and engulfment by macrophages, leading to foam cell formation and lesion development 2, 2, 3, 3 . How circulating LDL enters the artery wall to instigate atherosclerosis is unknown. Here we show in mice that scavenger receptor, class B type 1 (SR-B1) in endothelial Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Ankyrins are multifunctional adaptors that link speci®c proteins to the membrane-associated, spectrin± actin cytoskeleton. The N-terminal,`membrane-binding' domain of ankyrins contains 24 ANK repeats and mediates most binding activities. Repeats 13±24 are especially active, with known sites of interaction for the Na/K ATPase, Cl/HCO 3 anion exchanger, voltagegated sodium channel, clathrin heavy chain and L1 family cell adhesion molecules. Here we report the crystal structure of a human ankyrinR construct containing ANK repeats 13±24 and a portion of the spectrin-binding domain. The ANK repeats are observed to form a contiguous spiral stack with which the spectrin-binding domain fragment associates as an extended strand. The structural information has been used to construct models of all 24 repeats of the membrane-binding domain as well as the interactions of the repeats with the Cl/HCO 3 anion exchanger and clathrin. These models, together with available binding studies, suggest that ion transporters such as the anion exchanger associate in a large central cavity formed by the ANK repeat spiral, while clathrin and cell adhesion molecules associate with speci®c regions outside this cavity. Keywords: anion exchanger/ANK/ankyrin/clathrin/ spectrin IntroductionAnkyrins are protein adaptors that bridge between the spectrin±actin cytoskeleton and proteins involved in ion transport, cell adhesion and membrane traf®cking. Linkages with ion transporters represent the largest class and include associations with the Cl/HCO 3 anion exchanger, the Na/Ca exchanger, Na/K ATPase, IP3 receptor, ryanodine receptor and voltage-gated Na channels (Bennett and Stenbuck, 1979;Nelson and Veshnock, 1987;Srinivasan et al., 1988;Joseph and Samanta, 1993;Li et al., 1993;Bourguignon et al., 1995;Festy et al., 2001). These linkages serve to anchor the spectrin±actin cytoskeleton on the membrane and to concentrate these transporters in specialized membrane domains (reviewed in Bennett and Baines, 2001). The ankyrin linkages with cell adhesion molecules such as the L1 family (Davis et al., 1993) enhance cell adhesion and facilitate the incorporation of cells into tissues (Hortsch et al., 1998;More et al., 2001). Ankyrins also bind clathrin and participate in membrane traf®cking at the level of both coated pit budding (Michaely et al., 1999) and traf®cking of speci®c membrane proteins (Tuvia et al., 1999).Metazoan cells employ a large number of ankyrin isoforms for these different functions. Vertebrates have three ankyrin genes (ank1±3) that code for three families of proteins (ankyrinR, B and G, respectively). Most ankyrin isoforms contain two conserved domains that mediate protein±protein interactions. A central,`spectrinbinding' domain associates with b-spectrin, while the N-terminal,`membrane-binding' domain binds most other proteins. Isoform speci®city is determined by the variably spliced C-terminal domain, which contains isoformspeci®c targeting information and regulatory functions that modulate the binding activities of the two protein ...

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Rac1 and RhoA regulate membrane ruffling and stress fiber formation. Both molecules appear to exert their control from the plasma membrane. In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface. We now report that endogenous Rac1 and RhoA also have a polarized distribution at the cell surface. Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell. Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Rac1 and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA. We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae. Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes. The shift in location was not caused by inactivation of the RhoA effector domain. Moreover, chimeric proteins containing the C-terminal consensus site for Rac1 and RhoA prenylation were constitutively targeted to caveolae fractions. These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.The Rho family of small GTPases, which include Rac1, Cdc42, and RhoA, regulate the rearrangement of actin cytoskeleton when cells are exposed to growth factors and cytokines (1). A remarkable feature of these proteins is the ability of each family member to control a specific organizational state of the actin cytoskeleton in response to a common stimulus. For example, stimulation of fibroblasts with epidermal growth factor (EGF) 1 or platelet-derived growth factor (PDGF) activates Ras, Rac1, and then RhoA, suggesting that a G-protein cascade might be involved (2, 3). The activated Rac1 promotes ruffling of the plasma membrane, while the activated RhoA regulates the formation of stress fibers and focal adhesion sites. Lysophosphatidic acid (LPA), by contrast, activates RhoA but not Rac1, so stress fiber formation occurs but membrane ruffling does not (3). Stress fiber formation is also much faster in response to LPA than PDGF, suggesting that the timing of RhoA activation is an important control point for cytoskeleton reorganization.Rho family G-proteins function as molecular switches that promote the activity of effector molecules in response to exchanging GDP for GTP. As with the Ras family of G-proteins, multiple molecular interactions regulate the nucleotide state of the Rho family members, including GDP-GTP exchange factors that facilitate GTP loading and GTPase-activating proteins that stimulate GTP hydrolysis (4). The exchange process for the Rho family members, however, has an additional layer of regulation. Most GDP-bou...

The 24 ANK repeats of the membrane-binding domain of ankyrin form four folded subdomains of six ANK repeats each. These four repeat subdomains mediate interactions with at least seven different families of membrane proteins. In the erythrocyte, the main membrane target of ankyrin is the Cl -/HCO -3 anion exchanger. This report presents the first evidence that ankyrin contains two separate binding sites for anion exchanger dimers. One site utilizes repeat subdomain two (repeats 7-12) while the other requires both repeat subdomains three and four (repeats 13-24). The two sites are positively coupled with a Hill coefficient of 1.4. Since the anion exchanger exists as a dimer in the membrane, the presence of two binding sites on ankyrin allows ankyrin to interact with four anion exchangers simultaneously. These findings provide a direct demonstration of the versatility of ANK repeats in protein recognition, and have important implications for the organization of ankyrin-linked integral membrane proteins in erythrocytes as well as other cells.The spectrin-based membrane skeleton is an interlocking network of proteins which underlies the plasma membrane. The skeleton was first identified in erythrocytes, but it is also present under specialized regions of plasma membrane of cells in many tissues. The membrane skeleton is comprised of heterotetramers of α-and β-spectrin which form multiple, long range cross-links between cortical actin filaments. This structure is then linked to the membrane primarily by ankyrin, which possesses binding sites for β-spectrin and at least seven membrane proteins (1-2).In human erythrocytes, the interaction between ankyrin and the cytoplasmic domain of the Cl -/HCO -3 anion exchanger (Band 3) provides a major linkage between the spectrin skeleton and the plasma membrane (3-5). This linkage gives the erythrocyte membrane elastic properties which allows the plasma membrane to deform without vesiculation. Mutations or reductions in ankyrin which disrupt the linkage with the anion exchanger decouple the structural support of the spectrin skeleton from the membrane and result in severe hemolytic spherocytosis and anemia (2, 6, 7).The anion exchanger binding activity has been localized to the N-terminal, 89-kDa domain of ankyrin (8). The amino acid sequence of the 89-kDa domain is dominated by a tandem array of 24 ANK repeats: a 33-amino acid motif utilized in a diverse group of proteins for protein recognition (2, 9, 10). A 43-kDa proteolytic fragment derived from the C-terminal half of the 89-kDa domain is capable of interacting with the anion exchanger with high affinity, indicating that a major site of interaction is localized to repeats 13-24 (8). Activities of a series of deletion constructs demonstrate that repeats 22 and 23 are necessary but not sufficient for high affinity binding of the 43-kDa domain with the anion exchanger (11). It was not established in this study whether repeats 22 and 23 are required for stable folding or are involved in direct contact with the anion exchanger. M...