Hormonally regulated phosphoprotein of turkey erythrocytes: localization to plasma membrane

The catecholamine-stimulated cotransport of sodium and potassium ions across the plasma membrane of the turkey erythrocyte was previously found to be associated with increased 32P incorporation into a high molecular weight protein. To determine the subcellular localization of this phosphorylated protein, which we have termed goblin, a new method has been developed for isolation of pure plasma membranes from turkey erythrocytes. With this method, it has been demonstrated that goblin is located in the plasma membrane. Goblin is not extracted by solutions of low or high ionic strength but is partially extracted by nonionic detergents, indicating that it is not a component of turkey erythrocyte spectrin and suggesting that it may be an intrinsic protein of the plasma membrane. The data are compatible with a possible role for goblin in the hormonal control of ion movements across the plasma membrane.     

Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes

Phosphorylation by [gamma-32P]ATP of proteins associated with the plasma membrane of Saccharomyces cerevisiae has been studied both in vivo and in vitro. Although at least nine proteins are labeled in vivo, there is only one major protein labeled in vitro. This species with an apparent molecular weight of 114,000 has been identified as the plasma membrane Mg2+-ATPase. Phosphorylation of this enzyme occurs exclusively on serine residues. This is the first report that the proton-translocating ATPase of fungal plasma membranes is subject to phosphorylation by a protein kinase.     

Human ABCB6 localizes to both the outer mitochondrial membrane and the plasma membrane

Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.     

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase. Gel electrophoresis permitted the identification of a major protein component of the presynaptic membrane fraction which had a molecular weight of 67,000. This protein was not found in postsynaptic membrane or synaptic vesicle fractions. Thus it appeared to be specific to the nerve terminal plasma membrane.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Microscopy approaches to investigate protein dynamics and lipid organization

The structure of cell membranes has been intensively investigated and many models and concepts have been proposed for the lateral organization of the plasma membrane. While proteomics and lipidomics have identified many if not all membrane components, how lipids and proteins interactions are coordinated in a specific cell function remains poorly understood. It is generally accepted that the organization of the plasma membrane is likely to play a critical role in the regulation of cell function such as receptor signalling by governing molecular interactions and dynamics. In this review we present different plasma membrane models and discuss microscopy approaches used for investigating protein behaviour, distribution and lipid organization.     

Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases)

Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings.     

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.     

Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase

Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I-Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I-glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma Membrane Protein Clusters Appear in CFTR-Expressing Xenopus Laevis Oocytes after cAMP Stimulation

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per μm². Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm² while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per μm² while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins. Received: 11 September 2000/Revised: 13 December 2000     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

G proteins in cellular control.

In the past year, cDNA cloning has revealed substantial diversity in G protein alpha, beta and gamma subunits. The number of cellular functions recognized to be controlled by G proteins is also increasing. Most G proteins are associated with the cytoplasmic surface of the plasma membrane, and molecular mechanisms for membrane association of certain G protein subunits have been defined recently. Mutations in G protein subunits, both artificially induced and naturally occurring, have provided important insights into G protein structure and function.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts.

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [gamma-32P]ATP and [gamma-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occurred at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min. The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phosphorylation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular weight of about 100 000 and the ATP-specific main component a molecular weight of 110 000. The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.     

Calcium binding proteins in smooth muscle plasma membrane

Rat myometrium plasma membrane showed a number of 45Ca-binding proteins as identified by gel electrophoresis. An attempt was made to identify these either by studying the inhibition of this binding by several ions or by studying binding of these proteins to calmodulin, A9 an antibody against skeletal muscle Ca-binding proteins and Stains-all. On the basis of the molecular weight, calmodulin binding and La-sensitivity of Ca-binding, the Ca-binding protein at 137 +/- 2 kDa has been identified as the Ca-pump. This protein as judged from Coomassie blue staining forms a very small percentage of the proteins present in the plasma membrane.