Gene A. Scarborough

Three-dimensional map of the plasma membrane H+-ATPase in the open conformation.

The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cells plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200 mV (refs 1–3). Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 Å, this map reveals ten membrane-spanning α-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands.

Antiestrogens and steroid hormones: Substrates of the human P-glycoprotein

Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp). Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp. Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil. Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively. Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone. Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil. These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.

Drug-stimulated ATPase activity of the human P-glycoprotein

The human multidrug resistance protein, or P-glycoprotein (Pgp), exhibits a high-capacity drug-dependent ATP hydrolytic activity that is a direct reflection of its drug transport capability. This activity is readily measured in membranes isolated from cultured insect cells infected with a baculovirus carrying the humanmdrl cDNA. The drug-stimulated ATPase activity is a useful alternative to conventional screening systems for identifying high-affinity drug substrates of the Pgp with potential clinical value as chemosensitizers for tumor cells that have become drug resistant. Using this assay system, a variety of drugs have been directly shown to interact with the Pgp. Many of the drugs stimulate the Pgp ATPase activity, but certain drugs bind tightly to the drug-binding site of the Pgp without eliciting ATP hydrolysis. Either class of drugs may be useful as chemosensitizing agents. The baculovirus/insect cell Pgp ATPase assay system may also facilitate future studies of the molecular structure and mechanism of the Pgp.

2-D STRUCTURE OF THE NEUROSPORA CRASSA PLASMA MEMBRANE ATPASE AS DETERMINED BY ELECTRON CRYOMICROSCOPY

Large, well‐ordered 2‐D crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)‐ATPase grow rapidly on the surface of a polyethylene glycol‐containing mixture similar to that originally developed for growing 3‐D crystals of this integral membrane transport protein. Negative stain electron microscopy of the crystals shows that many are single layers. Cryoelectron microscopy of unstained specimens indicates that the crystals have a p6 layer group with unit cell dimensions of a = b = 167 A. Image processing of selected electron micrographs has yielded a projection map at 10.3 A resolution. The repeating unit of the ATPase crystals comprises six 100 kDa ATPase monomers arranged in a symmetrical ring. The individual monomers in projection are shaped like a boot. These results provide the first indications of the molecular structure of the H(+)‐ATPase molecule. They also establish the feasibility of precipitant‐induced surface growth as a rapid, simple alternative to conventional methods for obtaining 2‐D crystals of the integral membrane proteins useful for structure analysis.

A hexameric form of the Neurospora crassa plasma membrane H+-ATPase

As isolated by our recently developed large-scale procedure, the Neurospora plasma membrane H+-ATPase exists as a homogeneous, oligomeric complex of 105,000-Da monomers with a molecular mass equivalent to a spherical protein of about 1 million Da, as judged by its behavior during chromatography on calibrated columns of Sepharose CL-6B and CL-4B. Treatment of this complex with the nonionic detergent, Tween 20, followed by Sepharose column chromatography in the presence of this detergent produces particles with an apparent molecular mass reduced by 100-300 kDa, and, importantly, when the isolated complex is treated with Tween 20 and then subjected to Sepharose chromatography in the absence of detergent, fully viable, largely detergent-free, homogeneous particles with a molecular mass equivalent to a spherical protein of 670,000 Da are formed. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, treatment of the particles isolated in the presence of Tween 20 with glutaraldehyde progressively yields dimers, trimers, tetramers, pentamers, and hexamers of the 105,000-Da monomer, with the expected precursor-product relationships, but no species larger than a hexamer is formed. These results thus strongly indicate that these particles are hexamers of 105,000-Da monomers. Glutaraldehyde crosslinking experiments with the ca. 1 million- and 670,000-Da particles indicate that they too are hexamers, suggesting that the differences in the apparent sizes of the three types of particles are most likely due to bound detergents. Possible implications of these findings are discussed.

Structure and function of the P-type ATPases

The P-type ATPases are integral membrane proteins that generate essential transmembrane ion gradients in virtually all living cells. The structures of two of these have recently been elucidated at a resolution of 8 A. When considered together with the large body of biochemical information that has accrued for these transporters and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by these proteins.

Structure of the P-type ATPases.

Electron cryocrystallography of precipitant-induced two-dimensional surface crystals of the neurospora plasma membrane H+ - ATPase and tubular crystals of the sarcoplasmic reticulum Ca(2+)-ATPase has recently yielded structure maps for these ion transporters at a resolution of about 8 A. The membrane-embedded regions of these closely related enzymes are similar, but the cytoplasmic regions appear to be significantly different.

Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast

Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.

Large-scale isolation of the Neurospora plasma membrane H+-ATPase.

A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O2 to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H+-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with alpha-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H+-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 mumol of P1 released/min/mg of protein at 30 degrees C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.

An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein☆

A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required.