The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.
Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% a-helix and 17% bsheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 % 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 % 0.03 and 12.5 % 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V 1 without C from Manduca sexta resulted in a V 1 -Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.
Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.
The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V 1 and V O sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was puri¢ed to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel ¢ltration analysis and small-angle X-ray scattering, was approximately 28 þ 2 kDa, indicating that this protein is dimeric. With a radius of gyration (R g ) and a maximum size (D max ) of 2.7 þ 0.2 nm and 8.0 þ 0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G 38À144 form (the carboxyl-terminus) was expressed and puri¢ed. G 38À144 is homogeneous, with a molecular mass of approximately 12 þ 3 kDa, indicating a monomeric form in solution. ß
The effect of the ATPase activity of Manduca sexta V 1 ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V 1 were studied. The Vacuolar ATPases (V 1 V O ATPases) define an ubiquitous class of proton pumps, which utilize ATP hydrolysis to maintain an acidic pH inside the vacuole (1). The electrochemical ion gradient created across the vacuolar membrane is used for the accumulation of positively charged substrates such as calcium and basic amino acids (2). In addition to this storage function, the vacuolar compartment has secretory and proteolytic functions (3, 4). The V-ATPases, consisting of at least thirteen distinct subunits (A 3 :B 3 :C:D:E:F:G y :H z :a:d:c:cЈ:cЉ) are morphologically subdivided in two components: a membrane-bound domain, V O , that contains the ion channel, and an extrinsic domain, V 1 , in which ATP hydrolysis takes place (4, 5). The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8,9), revealed a molecule with a single, compact stalk.The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule. The x-ray data define a mushroom-shaped V 1 ATPase, which consists of an ϳ145 Å headpiece, joined by an elongated stalk (8). Image processing of electron micrographs of negatively stained V 1 (9, 12, 13) has revealed that the headpiece consists of a pseudo-hexagonal arrangement of six masses surrounding a seventh mass. These barrel-shaped masses of approximately 30 Å in diameter and 80 Å in length, which consist of the major subunits A and B, are arranged in an alternating manner (9). The hexagonal barrel of subunits A and B encloses a cavity of ϳ32 Å in which part of the central stalk is asymmetrically located. The stalk protrudes from the bottom side of the headpiece forming an angle of ϳ7°with the vertical axis of the molecule. At the upper end of the hexagonal barrel extensions can be observed, assumed to belong to the N termini of subunit A (9, 13). Further insights into the topology of the M. sexta V 1 ATPase were obtained by differential protease sensitivity, release by chaotropic agents (13), and cross-linking studies (13,14). These studies resulted in a model in which the subunits H, C, D, G, and F are exposed in the enzyme, whereas subunit E is shielded in the complex (6,13,14).Here we report an investigation of the structure-function relationship of the V 1 stalk...
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