Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10?nm disc-like structure ideally suited to purification and further biochemical study.     

The phosphorylation and isolation of two erythrocyte membrane proteins in vitro

Two proteins of bovine erythrocyte ghost membrane have been phosphorylated with γ-³²P-ATP and isolated by SDS polyacrylamide gel electrophoresis. One of the two proteins (MW 98,000) has been identified here as the phosphorylated intermediate of the Na⁺ + K⁺ activated ATPase. The other phosphorylated protein (MW 220,000) is apparently unrelated to the Na-K ATPase, but may be involved in other energy requiring membrane processes.     

The cyclic nucleotide-dependent phosphorylation of aortic smooth muscle membrane proteins

Membrane proteins of Mr 240,000, 130,000, and 85,000 (GS-proteins) were rapidly and selectively phosphorylated in particulate fractions of rabbit aortic smooth muscle in the presence of [Mg-32P]ATP and low concentrations of cGMP (Ka = 0.01 microM) or cAMP (Ka = 0.2 microM). The effects of both cyclic nucleotides in this preparation were mediated entirely by an endogenous, membrane-bound form of cGMP-dependent protein kinase (G-kinase). The GS-proteins were also phosphorylated by the soluble form of G-kinase purified from bovine lung; this effect was most evident following removal of endogenous G-kinase from the membranes using Na2CO3 and high salt washes. The membrane-bound and cytosolic forms of G-kinase phosphorylated the Mr 130,000 GS-protein with the same specificity as determined by two-dimensional peptide mapping. Despite this functional homology between the two forms of G-kinase, only the particulate enzyme appears to play a role in phosphorylating the GS-proteins. Although little endogenous cAMP-dependent protein kinase (A-kinase) activity was detected in washed aortic smooth muscle membranes, the GS-proteins could be phosphorylated when purified A-kinase catalytic subunit was added to this preparation. Peptide mapping of the Mr 130,000 GS-protein indicated that A-kinase phosphorylated a subset of the same peptides labeled by the two forms of G-kinase. The endogenous A-kinase of rabbit aortic smooth muscle homogenates was also found to phosphorylate the GS-proteins. Since the intracellular concentrations of cGMP or cAMP can be selectively elevated by different stimuli, these results suggest several possible mechanisms by which the phosphorylation state of the GS-proteins may be regulated by cyclic nucleotides: activation of the membrane-bound G-kinase by cGMP or cAMP; and activation of cytosolic A-kinase by cAMP.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

Polarized targeting of peripheral membrane proteins in neurons

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

The phosphorylation of the major proteins of the human erythrocyte membrane.

Both the major sialoglycoprotein (PAS-1) and the component designated by Fairbanks et al. (G. Fairbanks, T. L. Steck, and D. F. H. Wallach, 1971, Biochemistry10, 2606–2617) as Band 3 are shown to be bonafide phosphoproteins by virtue of the presence of covalently bound serine and threonine phosphate residues. In agreement with the findings of others, PAS-1 does not seem to be phosphorylated when ghosts are incubated with [γ-³²P]ATP, but the phosphorylation is significant (about 0.15 mol/mol) when the cells are incubated in the presence of ³²P_i. Band 3 is phosphorylated to the extent of 0.90 mol/mol, and these sites are apparently distributed in several places along the polypeptide chain. Spectrin is also a phosphoprotein containing approximately four molecules of phosphate per 450,000 daltons of protein. The phosphorylation of these three polypeptides is not stimulated by the presence of cAMP.     

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of 10–15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.     

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.     

Preferential turnover of membrane proteins in the intact Chlamydomonas flagellum

Radioactive labeling studies demonstrate a continuous incorporation of newly synthesized proteins and glycoproteins into the intact flagella of Chlamydomonas. This apparent turnover is preferentially occurring for membrane components. In particular, two classes of flagellar membrane components, one a high molecular weight (HMW) group of closely migrating glycoproteins and the other a protein with a MW around 65 kD, are continuously turning over in the vegetative cell. This selective protein turnover may explain the ability of Chlamydomonas to rapidly recover from proteolytic modification of the flagellar surface and to change its flagellar surface properties during the early events in mating.     

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.     

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.     

Evidence for the participation of cytosolic protein kinases in membrane phosphorylation in intact erythrocytes.

The effects of adenosine 3':5'-monophosphate (cyclic AMP) on the phosphorylation of membrane proteins in intact rabbit and human erythrocytes were investigated. The addition of cyclic AMP to intact human or rabbit erythrocytes results in an increase in the incorporation of ortho[32P]phosphate into several membrane protein components which are known to serve as substrates for the cyclic-AMP-dependent protein kinases. Thus this increase in protein phsophorylation is probably due to the activation of either soluble or membrane-bound cyclic-AMP-dependent protein kinases. Incubation of human erythrocytes in the presence of ortho [32P]phosphate and cyclic AMP also leads to the phosphorylation of a membrane protein component, band 7, which has not been previously detected in the autophosphorylation of isolated ghosts. Since rabbit erythrocyte membranes do not contain any cyclic-AMP-dependent protein kinase, the results suggest that cytoplasmic kinases also play a role in the phosphorylation of membrane proteins in intact cells.     

Short-time phosphorylation of plasma membrane proteins by endogenous kinases.

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence and absence of cyclic nucleotides. Using low concentrations of membrane protein the kinetics of ATP-dependent ³²P-incorporation showed a rapid phosphorylation reaction up to 2 sec of incubation which was stimulated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. This phenomenon was dependent on protein concentration.     

Difference in phosphorylation of neurofilament proteins in motor neurons and spinal ganglion cells

The composition of the neurofilament proteins (NFPs) in neuronal perikarya was examined by two-dimensional (2-D) gel electrophoresis of isolated perikarya of bovine spinal motor neurons. The extent of phosphorylation of the high molecular weight subunit of NFP (NFP-H) was compared between motor and sensory neuronal perikarya in spinal cord and spinal ganglion by immunocytochemistry with monoclonal antibodies (MAbs) to NFP. Of the 23 MAbs used in this study, one MAb (82E10) was specific to the highly phosphorylated component of NFP-H examined by 2-D immunoblot whereas another MAb (3A8) was specific to NFP-H irrespective of its level of phosphorylation. Immunocytochemically, 82E10 did not stain the perikarya of bovine and rabbit spinal motor neurons but 3A8 stained the perikarya in both animal species. These findings are consistent with 2-D immunoblot of neuronal perikarya of bovine motor neurons isolated in bulk. As for the spinal ganglia, 82E10 stained many, but not all, perikarya of sensory neurons of both animal species. These results indicate that the extent of phosphorylation of NFP-H in the perikarya of most spinal ganglion cells is higher than that of motor neurons. These findings suggest that the rate of phosphorylation of NFP-H in perikarya or the axonal transport of NFP from perikarya to proximal axons is uniform in spinal motor neurons but variable in spinal ganglion cells.     

Effect of ionizing radiation on the functional state of snail neurons. Cyclic nucleotide levels and phosphorylation of membrane proteins

A study was made of the effect of ionizing radiation, phospholipase A2, and low concentrations of mediators (for instance, acetylcholine and gamma-aminobutyric acid) on the content of cyclic nucleotides and phosphorylation of membrane proteins of Helix pomatia nervous ganglia. Ionizing radiation was shown to decrease considerably the levels of cAMP and cGMP which correlated with the diminution of membrane phosphorylation. Phospholipase A2 and low doses of mediators produced a modifying effect on the cyclic nucleotide content.     

The role of calmodulin in opioid-induced changes in the phosphorylation of rat striatal synaptic membrane proteins

Chronic morphine treatment of rats decreased the level of phosphorylation of synaptic membrane proteins of the striatum assayed in vitro. Although the patterns of phosphorylated proteins separated on SDS-gel electrophoresis from morphine-tolerant rats resembled patterns produced by lowering Ca²⁺ levels in the assay, supplementation of the protein kinase assay with Ca²⁺ and its binding protein, calmodulin, did not restore full kinase activity. The addition of methadone or etorphine to the protein kinase in vitro however, was able to block the Ca²⁺-calmodulin stimulation of phosphorylation in both synaptic membranes and intact synaptosomes. These data suggest that opioids produce an irreversible (or slowly reversible) defect in the Ca²⁺-dependent protein kinase system of striatal membranes.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.