Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p, a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain.

Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species. IPC-C, which is the predominant form found in wild-type cells. Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C26-fatty acid. Elimination of the SCS7 gene suppresses the Ca(2+)-sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca(2+)-sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b5-like domain and a domain that resembles the family of cytochrome b5-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C26-fatty acid of IPC-C.

SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of inositolphosphorylceramide.

Saccharomyces cerevisiae cells require two genes, CSG1/SUR1 and CSG2, for growth in 50 mM Ca2+, but not 50 mM Sr2+. CSG2 was previously shown to be required for the mannosylation of inositolphosphorylceramide (IPC) to form mannosylinositolphosphorylceramide (MIPC). Here we demonstrate that SUR1/CSG1 is both genetically and biochemically related to CSG2. Like CSG2, SUR1/CSG1 is required for IPC mannosylation. A 93-amino acid stretch of Csg1p shows 29% identity with the alpha-1, 6-mannosyltransferase encoded by OCH1. The SUR1/CSG1 gene is a dose-dependent suppressor of the Ca(2+)-sensitive phenotype of the csg2 mutant, but overexpression of CSG2 does not suppress the Ca2+ sensitivity of the csg1 mutant. The csg1 and csg2 mutants display normal growth in YPD, indicating that mannosylation of sphingolipids is not essential. Increased osmolarity of the growth medium increases the Ca2+ tolerance of csg1 and csg2 mutant cells, suggesting that altered cell wall synthesis causes Ca(2+)-induced death. Hydroxylation of IPC-C to form IPC-D requires CCC2, a gene encoding an intracellular Cu2+ transporter. Increased expression of CCC2 or increased Cu2+ concentration in the growth medium enhances the Ca2+ tolerance of csg1 mutants, suggesting that accumulation of IPC-C renders csg1 cells Ca2+ sensitive.

Sequence, mapping and disruption of CCC2, a gene that cross-complements the Ca(2+)-sensitive phenotype of csg1 mutants and encodes a P-type ATPase belonging to the Cu(2+)-ATPase subfamily.

We have isolated, sequenced, mapped and disrupted a gene, CCC2, from Saccharomyces cerevisiae. This gene displays non-allelic complementation of the Ca(2+)-sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P-type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca(2+)-induced death of csg1 mutant cells is related to Cu2+ metabolism. Cells without CCC2 require increased Cu2+ concentrations for growth. Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess Cu2+.

Phosphate, nitrendipine and valinomycin increase the Ca2+/ATP coupling ratio of rat skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase.

Nitrendipine and valinomycin act synergistically to stimulate ATP-dependent Ca2+ accumulation by rat skeletal muscle sarcoplasmic reticulum vesicles 3-fold. The stimulation is not caused by activation of the Ca(2+)-ATPase or by inhibition of the sarcoplasmic reticulum Ca2+ channel, but is due to an increased efficiency of transport by Ca(2+)-loaded vesicles. At low Ca2+ concentrations, nitrendipine+valinomycin inhibits Ca2+ uptake by increasing the Ca2+ KM but does not effect equilibrium Ca2+ binding to the Ca(2+)-ATPase (Kd = 0.75 microM). In the presence of 50 mM phosphate, nitrendipine+valinomycin increases the steady-state coupling ratio (Ca2+ accumulated per ATP hydrolyzed) from 0.6 to 1.9 by decreasing the rate of ATP hydrolysis by 72%, while reducing the Ca2+ accumulation rate by only 13%. The rates of both passive and Ca(2+)-ATPase-mediated Ca2+ release are reduced by nitrendipine+valinomycin. The data indicate that nitrendipine and valinomycin act directly on the Ca(2+)-ATPase to decrease the ATP hydrolysis rate, increase the Ca2+ KM, decrease Ca2+ efflux, and increase the Ca2+/ATP coupling ratio of Ca(2+)-loaded vesicles.

Activation and inhibition of the sarcoplasmic reticulum Ca2+ channel by the polycationic dyes Hoechst 33342 and Hoechst 33258.

The polycationic dyes, Hoechst 33342 (Bisbenzimide,2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H- benzimidazole) and Hoechst 33258 (Bisbenzimide,2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)- 2,5'-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca2+ channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca2+ efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca2+ channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca2+ channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca2+ movement through the channel, whereas the hydroxy group only reduces the rate of Ca2+ movement.

Activation of Ca2+ release from sarcoplasmic reticulum vesicles by 4-alkylphenols.

4-Alkylphenols induce Ca2+ release from junctional (terminal cisternae) sarcoplasmic reticulum vesicles, but not from nonjunctional sarcoplasmic reticulum vesicles. The 4-alkylphenol concentration required to induce Ca2+ release decreases about threefold for every methylene carbon increase in the alkyl chain length, indicating that the Ca(2+)-releasing potency of 4-alkylphenols is related to their ability to partition into the membrane. The rate and amount of Ca2+ release induced by relatively low 4-octylphenol concentrations (25 nmol/mg protein) are altered by the sarcoplasmic reticulum Ca2+ channel activators, Ca2+ and ATP, and the Ca2+ channel inhibitors, Mg2+ and ruthenium red. Ca2+ release induced by 250 nmol 4-octylphenol/mg protein is much less influenced by Ca2+ channel activators and inhibitors; however, even at this high 4-octylphenol concentration, Ca2+ release is not induced from nonjunctional sarcoplasmic reticulum vesicles. The data indicate that 4-alkylphenols induce Ca2+ release by activating the sarcoplasmic reticulum Ca2+ channel.

Mechanism of magainin 2a induced permeabilization of phospholipid vesicles.

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)-induced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages--an initial rapid phase, with t1/2 approximately 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptide-lipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a.

Biogenesis of transverse tubules in skeletal muscle in vitro.

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.

Secondary structure of calsequestrin in solutions and in crystals as determined by Raman spectroscopy.

Calsequestrin has been precipitated with calcium into five different crystal forms: cruciform twins, flat rectangles, thin needles, bipyramids, rectangular prisms, and a sixth precrystalline form, spheres. Raman spectra of the spheres and the cruciform twins are the same. The Raman spectrum of a physiological concentration (10%) of calsequestrin in calcium-free solution is the same as the spectrum of calcium precipitated calsequestrin in the amide I region, and in the C-C stretching region, but these spectra are different in the amide III region. The Raman spectrum of unfolded calsequestrin in 5 M guanidine hydrochloride is quite different from the other spectra, but it is not similar to the spectra of other unfolded proteins. Estimates of secondary structure from the amide I region indicate that calsequestrin in calcium-free solution and calcium-precipitated forms has 40 +/- 5% helix, 30 +/- 4% beta-strand, and 18 +/- 2% reverse turn. Secondary structure estimates calculated from the amide III region are not significantly different. They indicate 41 +/- 5% helix and 36 +/- 6% beta-strand for the precipitated forms, and 32 +/- 5% helix and 39 +/- 6% beta-strand for solutions. Calsequestrin unfolded in 5 M guanidine hydrochloride at 100 mg/ml gives 24 +/- 5% helix and 48 +/- 6% beta-strand.

Comparison of the rat microsomal Mg-ATPase of various tissues.

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3'-diethyl-2,2'-thiadicarbocyanine (Di-S-C2(5] to monitor inside positive or inside negative membrane potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase.

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle.

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg2+-ATPase (5-25 mumol Pi/mg min). The rate of ATP hydrolysis by the Mg2+-ATPase was nonlinear but decayed exponentially (first-order rate constant greater than or equal to 0.2 s-1 at 37 degrees C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg2+-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg2+-ATPase to detergents. It is proposed that the regulation of the Mg2+-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.

The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca2+ transport. The absorbance and fluorescence responses of 3,3'-diethyl-2,2'-indodicarbocyanine and oxonol VI during ATP-dependent Ca2+ transport are influenced by the effect of accumulated Ca2+ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca2+ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca2+, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulum in vivo and in vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca2+ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca2+ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca2+ translocation. The potential generated during Ca2+ transport is rapidly dissipated by passive ion fluxes across the membrane.

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles.

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.

The binding of arsenazo III to cell components.

The Ca2+ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1--2 mM arsenazo III, the estimated free arsenazo III concentration is only 50--200 microM; 80--90% of the bound arsenazo III is associated with soluble proteins. The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca2+; this is due to a decrease in the affinity of the protein-bound dye for Ca2+. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca2+, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye. The Ca2+ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.