Topology prediction of membrane proteins.

A new method is described for prediction of protein membrane topology (intra- and extracellular sidedness) from multiply aligned amino acid sequences after determination of the membrane-spanning segments. The prediction technique relies on residue compositional differences in the protein segments exposed at each side of the membrane. Intra/extracellular ratios are calculated for the residue types Asn, Asp, Gly, Phe, Pro, Trp, Tyr, and Val, preferably found on the extracellular side, and for Ala, Arg, Cys, and Lys, mostly occurring on the intracellular side. The consensus over these 12 residue distributions is used for sidedness prediction. The method was developed with a test set of 42 protein families, for which all but one were correctly predicted with the new algorithm. This represents an improvement over predictions based on the widely used "positive-inside rule" and other techniques, where at least six mispredictions were observed for the same data set. Further, application of this and other methods to 12 protein families not in the test set still showed the better performance of the present technique, which was subsequently applied to another set of membrane protein families where the topology has yet to be determined.     

Curvature-mediated interactions between membrane proteins.

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

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.     

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.     

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.