Elucidation of the Stator Organization in the V-ATPase of Neurospora crassa

Venzke, David; Domgall, Ines; Köcher, Thomas; Féthière, James; Fischer, Susanne; Böttcher, Bettina

doi:10.1016/j.jmb.2005.04.033

Cited by 42 publications

(56 citation statements)

References 60 publications

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“…Further studies demonstrated that the stator structure, in fact, consisted of two peripheral stalks that are positioned at an angle of 120° around the complex (Ubbink-Kok et al 2000;Boekema et al 1999). Subsequently, similar observations were made with V-type and A-type ATPases from other sources by others Zhang et al 2003;Bernal and Stock 2004;Venzke et al 2005). A major challenge remains the localization of the diVerent subunits in the stalk regions.…”

Section: Introductionsupporting

confidence: 64%

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

et al. 2006

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The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na + pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and F, G) that have very similar mobilities on SDS-PAGE of the puriWed complex (24.3 and 22.7 kDa, and 12.3 and 11.6 kDa) was established by tryptic digestion of the protein bands followed by mass spectrometric analysis of the peptides.

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Section: Introductionsupporting

confidence: 64%

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

et al. 2006

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“…The resolution of this map, as estimated by the Fourier shell correlation of two maps calculated separately from two halves of the data set, was ϳ15 Å (Fig. 2D) (14,15).…”

Section: Resultsmentioning

confidence: 99%

“…However, faithful structures of a large number of protein complexes have been determined at medium resolution from images of negatively stained particles such as those we have analyzed (14,15,(25)(26)(27). This structural analysis of an intramembrane-cleaving protease reveals a globular architecture of the physiologically active ␥-secretase complex, with the long-axis in the plane of the membrane, a sizable interior chamber, and discrete openings to the extracellular and cytoplasmic spaces.…”

Section: Discussionmentioning

confidence: 99%

Electron microscopic structure of purified, active γ-secretase reveals an aqueous intramembrane chamber and two pores

Lazarov

Fraering²,

Ye³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

160

150

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␥-Secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N-and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid ␤-peptide (A␤) from its precursor, APP. ␥-Secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of A␤40 and A␤42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of ϳ20 -40 Å in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, ϳ120-Å-long complex within the membrane and revealed ϳ20-Å pores at the top and bottom that provide potential exit ports for cleavage products to the extra-and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.electron microscopy ͉ image analysis ͉ intramembrane protease

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“…The proton-translocating part of the enzyme, named Vo, is composed of six types of subunits embedded in the membrane, with a reported stoichiometry of a 1 c 4 -5 cЈ 1 cЉ 1 d 1 e 1 (1,4). ATP is hydrolyzed within the V1 part of the enzyme, a roughly globular structure connected to Vo by at least two stalks (19,20). V1 contains eight types of subunits with a reported stoichiometry of A 3 B 3 CDEFG 2 H 1-2 .…”

mentioning

confidence: 99%

A Model for the Proteolipid Ring and Bafilomycin/Concanamycin-binding Site in the Vacuolar ATPase ofNeurospora crassa

Bowman¹,

McCall²,

Baertsch³

et al. 2006

J. Biol. Chem.

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The vacuolar ATPase has been implicated in a variety of physiological processes in eukaryotic cells. Bafilomycin and concanamycin, highly potent and specific inhibitors of the vacuolar ATPase, have been widely used to investigate the enzyme. Derivatives have been developed as possible therapeutic drugs. We have used random mutagenesis and site-directed mutagenesis to identify 23 residues in the c subunit involved in binding these drugs. We generated a model for the structure of the ring of c subunits in Neurospora crassa by using data from the crystal structure of the homologous subunits of the bacterium Enterococcus hirae (Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G., and Walker, J. E. (2005) Science 308, 654 -659). In the model 10 of the 11 mutation sites that confer the highest degree of resistance are closely clustered. They form a putative drug-binding pocket at the interface between helices 1 and 2 on one c subunit and helix 4 of the adjacent c subunit. The excellent fit of the N. crassa sequence to the E. hirae structure and the degree to which the structural model predicts the clustering of these residues suggest that the folding of the bacterial and eukaryotic polypeptides is very similar. The vacuolar ATPase (V-ATPase)2 is a component of almost all the eukaryotic cell membranes derived from the endoplasmic reticulum, e.g. Golgi, vacuoles, and coated vesicles (1-4). It generates an electrochemical gradient for protons, providing a driving force for metabolite transport. It also plays a major role in adjusting the pH within organelles.In animals many diverse physiological processes depend on the function of the V-ATPase. For example, a single mutation of the enzyme in humans was shown to cause both loss of kidney function and deafness (5). The V-ATPase is critical for the growth of bone (6), and overexpression of the enzyme has been correlated with the metastasis of breast cancer cells (7). The common thread linking these physiological events is secretion of protons by specialized cells that have high amounts of V-ATPase in the plasma membrane. A high rate of proton transport by the V-ATPase appears to be a key element in the development of diseases such as osteoporosis and in the growth of certain types of tumors. These findings have led to an investigation of the use of V-ATPase inhibitors as therapeutic drugs (8, 9).Several different classes of natural products, such as the benzolactone enamides and the chondropsins, have been shown to be potent and specific inhibitors of V-ATPases (10 -12). The most widely used inhibitors are two similar macrolide antibiotics, bafilomycin and concanamycin. These molecules inhibit the V-ATPase from all eukaryotic cells that have been tested, typically with half-maximal inhibition at 0.1-5 nM when assayed in vitro (13-15). At higher concentrations these antibiotics also inhibit in vivo, and derivatives of bafilomycin were effective in treating osteoporosis in a rat model system (16).We have been trying to identify the site on the V-ATPase at which bafilomycin an...

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Elucidation of the Stator Organization in the V-ATPase of Neurospora crassa

Cited by 42 publications

References 60 publications

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

Electron microscopic structure of purified, active γ-secretase reveals an aqueous intramembrane chamber and two pores

A Model for the Proteolipid Ring and Bafilomycin/Concanamycin-binding Site in the Vacuolar ATPase ofNeurospora crassa

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