Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.     

Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER- specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.     

Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae

Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle. We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation. To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific. In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation. We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients. This procedure revealed a bimodal distribution of ER markers. The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively. In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml. To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I. Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation. Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.     

Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.     

Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein.

Oligosaccharyltransferase catalyzes the N-linked glycosylation of asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic reticulum (RER). A protein complex composed of 66, 63, and 48 kd subunits copurified with oligosaccharyltransferase from canine pancreas. The 66 and 63 kd subunits were shown by protein immunoblotting to be identical to ribophorin I and II, two previously identified RER glycoproteins that colocalize with membrane-bound ribosomes. The transmembrane segment of ribophorin I was found to be homologous to a recently proposed dolichol recognition consensus sequence. Based on a revision of the consensus sequence, we propose a model for the interaction of dolichol with the glycosyltransferases that catalyze the assembly and transfer of lipid-linked oligosaccharides.     

Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.     

Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage-dependent calcium channels.

GTP binding proteins have been proposed to play a role in excitation--contraction coupling. In a precedent study [Toutant et al., (1988), Biochem. J., 405-409], we determined that Bordetella pertussis toxin is able to catalyse ADP-ribosylation of two substrates in the detergent soluble fraction of total muscle extracts. Purified fractions of transverse tubule membranes (T-tubule membranes), a key element of the excitation--contraction coupling, were shown to exhibit a major ADP-ribosylated substrate at 40 kd and an immunoreactivity with antisera raised against purified bovine brain Go alpha or G beta. In the present study, we have investigated the cellular distribution of G protein subunits in comparison with that of the voltage-dependent Ca2+ channels by immunofluorescence on transverse and longitudinal sections of fast and slow muscles. With affinity-purified antibodies against G beta subunits, a fluorescent labelling underlined the myofibrils and sarcolemma, whereas a strong immunoreaction in a dotted pattern evoked the presence of the subunit in repetitive triadic structures. With anti-Go alpha antibodies, the immunofluorescence was more clearly focussed on a dotted pattern and the co-location with the voltage-dependent Ca2+ channel immunoreactivity indicates that both proteins were located in very close subcellular structures. Immunoblot analysis and PTX ADP-ribosylation of the purified light sarcoplasmic reticulum (LSR), heavy sarcoplasmic reticulum (HSR) and T-tubule subcellular fractions indicate the discrete presence of G proteins in LSR, an unambiguous labelling of the HSR fraction, while T-tubule membranes clearly appear very rich in a Go-like protein, confirming the observed preferential immunocytochemical distribution of G protein subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of endogenous mono-ADP-ribosylation of cardiac proteins via anti-ADP-ribosylarginine immunoreactivity

Arginine-specific mono-ADP-ribosylation of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosylpolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylation products of quail heart. Treatment of Immobilon-bound ADP-ribosylated Gs protein with hydroxylamine under conditions that remove ADP-ribose from its arginines eliminated R-28 immunoreactivity to Gs. Also, R-28 immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac Gi/Go proteins. The antiserum did not appear to react with ADP-ribosylasparagine of Rho (formed by C3 toxin), ADP-ribosyldiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosylarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest immunoreactivity in the sarcolemma with significant immunoreactivity in denser membrane fractions. The cytosol also contained an immunoreactive band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous immunoreactive bands contain ADP-ribosylarginine. In conclusion, a polyclonal antiserum that recognizes ADP-ribosylarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylation products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-ribosylated on arginines.     

Ribosome-independent regulation of translocon composition and Sec61alpha conformation

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.     

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.     

Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform

The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. All subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes. Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin. Phosphorylation studies in the absence of Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.     

Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly-conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full-length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N-terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti-ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150-amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.     

Effects of a mechanical stimulation of localization of annexin-like proteins in Bryonia dioica internodes.

Mechanical stimulation exerted by rubbing a young internode of Bryonia dioica plants inhibits its growth. Previous cellular and biochemical studies showed that this growth inhibition is associated with Ca(2+) redistribution and profound modifications of plasma membrane characteristics. We extracted and purified Ca(2+)-dependent phospholipid-binding proteins from B. dioica internodes. Two main proteins, p33 and p35, and other minor bands were isolated and identified as annexin-like proteins because of their biochemical properties and their cross-reactions with antibodies against maize (Zea mays L.) annexins. Rabbit antiserum was obtained by injection of B. dioica p35. This antiserum was used for the immunocytolocalization of annexin-like proteins in internode parenchyma cells. It appeared that the distribution of annexin-like proteins was different before and 30 min after the mechanical stimulation. Western analysis of proteins in membrane fractions after separation by free-flow electrophoresis showed that p35 was present in most fractions, whereas p33 appeared mainly in plasmalemma-enriched fractions after the mechanical stimulation. It is hypothesized that a subcellular redistribution of these proteins might be involved in growth inhibition by mechanical stress.     

A monoclonal antibody to the Ca2+-ATPase of cardiac sarcoplasmic reticulum cross-reacts with slow type I but not with fast type II canine skeletal muscle fibers: an immunocytochemical and immunochemical study

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (II D8) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+-ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following beta-adrenergic stimulation.     

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.     

Distribution and localization of calmodulin-binding proteins in bull spermatozoa

Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca(2+). Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca(2+) in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.     

A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato

Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon Ca2+-ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots.