V-ATPases are ubiquitous proton pumps responsible for compartment acidification in all eukaryotic cells (1, 2). These pumps couple hydrolysis of cytosolic ATP to proton transport into the lysosome/vacuole, endosomes, Golgi apparatus, clathrin-coated vesicles, and synaptic vesicles. Through their role in organelle acidification, V-ATPases are linked to cellular functions as diverse as protein sorting and targeting, zymogen activation, cytosolic pH homeostasis, and resistance to multiple types of stress (3). They are also recruited to the plasma membrane of certain cells, where they catalyze proton export (4, 5).V-ATPases are evolutionarily related to ATP synthases of bacteria and mitochondria and consist of two multisubunit complexes, V 1 and V 0 , which contain the sites for ATP hydrolysis and proton transport, respectively. Like the ATP synthase (F-ATPase), V-ATPases utilize a rotational catalytic mechanism. ATP binding and hydrolysis in the three catalytic subunits of the V 1 sector generate sequential conformational changes that drive rotation of a central stalk (6 -8). The central stalk subunits are connected to a ring of proteolipid subunits in the V 0 sector that bind protons to be transported. The actual transport is believed to occur at the interface of the proteolipids and V 0 subunit a. Rotational catalysis will be productive in proton transport only if V 0 subunit a is held stationary, whereas the proteolipid ring rotates (8). This "stator function" resides in a single peripheral stalk in F-ATPases (9, 10), but is distributed among up to three peripheral stalks in V-ATPases (11-13). The peripheral stator stalks link V 0 subunit a to the catalytic headgroup and ensures that there is rotation of the central stalk complex relative to the V 0 a subunit and catalytic headgroup.Eukaryotic V-ATPases are highly conserved in both their overall structure and the sequences of individual subunits. Although homologs of most subunits of eukaryotic V-ATPases are present in archaebacterial V-ATPases (also known as A-ATPases), the C and H subunits are unique to eukaryotes. Both subunits have been localized at the interface of the V 1 and V 0 sectors, suggesting that they are positioned to play a critical role in structural and functional interaction between the two sectors (14 -16). The yeast C and H subunits are the only eukaryotic V-ATPase subunits for which x-ray crystal structures are available (17,18). The structure of the C subunit revealed an elongated "dumbbell-shaped" molecule, with foot, head, and neck domains (18). The structure of the H subunit indicated two domains. The N-terminal 348 amino acids fold into a series of HEAT repeats and are connected by a 4-amino acid linker to a C-terminal domain containing amino acids 352-478 (17). These two domains have partially separable functions in the context of the assembled V-ATPase (19). Complexes containing only the N-terminal domain of the H subunit (H-NT) 2 supported some ATP hydrolysis but little or no proton pumping in isolated vacuolar vesicles (19,20). The...

The H subunit of the yeast V-ATPase is an extended structure with two relatively independent domains, an N-terminal domain consisting of amino acids 1-348 and a C-terminal domain consisting of amino acids 352-478 (Sagermann, M., Stevens, T. H., and Matthews, B. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 7134 -7139). We have expressed these two domains independently and together in a yeast strain lacking the H subunit (vma13⌬ mutant). The N-terminal domain partially complements the growth defects of the mutant and supports ϳ25% of the wild-type Mg 2؉ -dependent ATPase activity in isolated vacuolar vesicles, but surprisingly, this activity is both largely concanamycin-insensitive and uncoupled from proton transport. The C-terminal domain does not complement the growth defects, and supports no ATP hydrolysis or proton transport, even though it is recruited to the vacuolar membrane. Expression of both domains in a vma13⌬ strain gives better complementation than either fragment alone and results in higher concanamycin-sensitive ATPase activity and ATP-driven proton pumping than the N-terminal domain alone. Thus, the two domains make complementary contributions to structural and functional coupling of the peripheral V 1 and membrane V o sectors of the V-ATPase, but this coupling does not require that they be joined covalently. The N-terminal domain alone is sufficient for activation of ATP hydrolysis in V 1 , but the C-terminal domain is essential for proper communication between the V 1 and V o sectors. V-ATPases2 are ATP-driven proton pumps responsible for acidification of intracellular organelles in all eukaryotic cells and for proton transport across the plasma membrane in certain cells (1, 2). V-ATPases are comprised of a peripheral complex containing the sites for ATP hydrolysis, the V 1 sector, attached to a membrane complex containing the proton pore, the V o sector (2). The yeast V-ATPase has proved to be an excellent model system for eukaryotic V-ATPases. In yeast, eight subunits, designated A, B, C, D, E, F, G, and H, make up the V 1 sector, and at least six subunits designated a, c, cЈ, cЉ, d, and e subunits make up the V o sector (3, 4). All of these subunits have homologues in higher eukaryotes, and in many cases, these homologues have been found to functionally substitute for each other.V-ATPases share a common evolutionary ancestor with F 1 F o -ATP synthases (5). The core of the catalytic machinery, specifically the ATP hydrolyzing A subunit, the B subunit, and proteolipid subunits (c, cЈ, and cЉ), show significant homology with ATP synthase subunits. However, V-ATPases are dedicated proton pumps, while ATP synthases operate primarily in the direction of ATP synthesis in vivo. The catalytic mechanism is fundamentally similar between these two types of enzymes and involves subunit rotation (6, 7). In ATP synthases, two stalk structures connect the peripheral headgroup containing the ATP binding subunits and the integral membrane complex containing the proton pore. These two structures act as a "roto...

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BackgroundApolipoprotein E (apoE) is a major cholesterol transport protein found in association with brain amyloid from Alzheimer's disease (AD) patients and the ε4 allele of apoE is a genetic risk factor for AD. Previous studies have shown that apoE forms a stable complex with amyloid β (Aβ) peptides in vitro and that the state of apoE lipidation influences the fate of brain Aβ, i.e., lipid poor apoE promotes Aβ aggregation/deposition while fully lipidated apoE favors Aβ degradation/clearance. In the brain, apoE levels and apoE lipidation are regulated by the liver X receptors (LXRs).ResultsWe investigated the hypothesis that increased apoE levels and lipidation induced by LXR agonists facilitates Aβ efflux from the brain to the cerebral spinal fluid (CSF). We also examined if the brain expression of major apoE receptors potentially involved in apoE-mediated Aβ clearance was altered by LXR agonists. ApoE, cholesterol, Aβ40, and Aβ42 levels were all significantly elevated in the CSF of rats after only 3 days of treatment with LXR agonists. A significant reduction in soluble brain Aβ40 levels was also detected after 6 days of LXR agonist treatment.ConclusionsOur novel findings suggest that central Aβ lowering caused by LXR agonists appears to involve an apoE/cholesterol-mediated transport of Aβ to the CSF and that differences between the apoE isoforms in mediating this clearance pathway may explain why individuals carrying one or two copies of APOE ε4 have increased risk for AD.

Extended chemical shift anisotropy amplification (xCSA) is applied for measuring (13)C/(15)N chemical shift anisotropy (CSA) of uniformly labeled proteins under magic-angle spinning (MAS). The amplification sequence consists of a sequence of π-pulses that repetitively interrupt MAS averaging of the CSA interaction. The timing of the pulses is designed to generate amplified spinning sideband manifolds which can be fitted to extract CSA parameters. The (13)C/(13)C homonuclear dipolar interactions are not affected by the π-pulses due to the bilinear nature of the spin operators and are averaged by MAS in the xCSA experiment. These features make the constant evolution-time experiment suitable for measuring CSA of uniformly labeled samples. The incorporation of xCSA with multi-dimensional (13)C/(15)N correlation is demonstrated with a GB1 protein sample as a model system for measuring (13)C/(15)N CSA of all backbone (15)NH, (13)CA and (13)CO sites.