Integral Membrane Polypeptide Research Articles

Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts.

Nonpenetrating proteolytic enzymes (such as thermolysin) were used to probe the cytosolic surface of the outer envelope membrane from spinach chloroplasts. Up to 20 different envelope polypeptides were susceptible to a mild digestion of isolated intact chloroplasts by thermolysin. Most of the thermolysin-sensitive envelope polypeptides were not extracted by a mixture of chloroform/methanol (2:1, v/v). A clear exception was E10 which is hydrophobic and, in addition, is an integral membrane polypeptide. Using antibodies to envelope polypeptides sensitive (E10 and E24) and insensitive (E30 and E37) to thermolysin, we demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, induced agglutination of intact chloroplasts. In addition, immunofluorescence experiments demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, gave a green fluorescence at the outer surface of intact chloroplasts. These experiments demonstrate that E10 and E24, and probably all the thermolysin-sensitive envelope polypeptides, are accessible from the cytosolic side of the outer membrane of the chloroplast envelope.

Lipid enrichment of thylakoid membranes. I. Using soybean phospholipids

(1) Using asolectin (mixed soybean phospholipids) liposomes, extra lipid, with or without additional plastoquinone, has been introduced into isolated thylakoid membranes of pea chloroplasts. (2) Evidence for this lipid enrichment was obtained from freeze-fracture which indicated that a decrease in the numbers of EF and PF particles per unit area of membrane occurred with increasing lipid incorporation. The decrease was not due to loss of integral membrane polypeptides as judged by assay of cytochrome present or SDS-polyacrylamide gel electrophoresis of lipid-enriched membrane fractions. Moreover, the enrichment procedure did not lead to extraction of low molecular weight lipophilic membrane components or of thylakoid membrane lipids. (3) The introduction of phospholipids into the membrane affected steady-state electron transport. Inhibition of electron transport was observed when either water (Photosystem (PS) II + PS I) or duroquinol (PS I) was used as electron donor with methyl viologen as electron acceptor, and the degree of inhibition increased with higher enrichment levels. Introduction of exogenous plastoquinone with the additional lipid had little effect on whole-chain electron transport, but caused an increase in the 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB)-sensitive rate of PS I electron transport. The inhibition was also detected by flash-induced oxidation-reduction changes of cytochrome f.

Comparisons of plasma membrane polypeptides from soybean and alfalfa

Abstract Polypeptides were solubilized with sodium dodecyl sulfate from plasma membrane vesicles of eight varieties of soybean roots [ Glycine max (L.) Merr.] and of cultured alfalfa cells ( Medicago sativa L.). The solubilized polypeptides were analysed by 2D-polyacrylamide gel electrophoresis. Apparent isoelectric point and MW values were obtained for 80 soybean plasma membrane polypeptides and 44 alfalfa plasma membrane polypeptides. From these data composite distribution patterns were constructed, which are representative of the soybean or alfalfa 2D-gels, respectively. The results showed that the general polypeptide staining patterns were similar for all the soybean varieties, but some minor differences were evident. The alfalfa electrophoretograms differed markedly from the soybean electrophoretograms in specific details, though some general pattern similarities were noted. The data are discussed in terms of a physiological role for the integral plasma membrane polypeptides and in terms of the potential for distinguishing among soybean varieties and between species at the plasma membrane polypeptide level.

A rabbit erythrocyte membrane protein associated with L-lactate transport.

Chemical Labeling has been employed to attempt to identify the lactate transport protein of rabbit erythrocytes, which have a very high capacity for stereoselective lactate transport. The lactate transport protein catalyzes lactate/proton cotransport (or lactate/hydroxyl exchange) at physiological pH, as demonstrated by uphill proton fluxes induced by lactate gradients. However, lactate/lactate and lactate/pyruvate exchange are considerably more rapid than lactate/proton cotransport. Although the lactate transporter is less sensitive to inhibition by the stilbenedisulfonate derivative H2DIDS (4,4'diisothiocyano-2,2'-dihydrostilbenedisulfonate) than is the inorganic anion exchanger (band 3), H2DIDS is nonetheless a reasonably potent inhibitor of the lactate transport. A 1-h treatment with 10(-4) M H2DIDS irreversibly inhibits lactate/lactate exchange by greater than 80%. This inhibition appears to be related to the labeling (by [3H]H2DIDS) of an integral membrane polypeptide of Mr = 43,000. This [3H]H2DIDS-labeled polypeptide is absent from human erythrocytes, which have a 100-fold lower Vmax for lactate transport than do rabbit erythrocytes. These experiments suggest that the polypeptide of Mr = 43,000 is a component of the lactate transport system.

A comparative study of the localization of wheat-germ agglutinin and its potential receptors in wheat grains

Wheat-germ agglutinin is located only in the embryo of a dry wheat seed and not in the endosperm tissue. This distribution remains unaltered for up to 96 h of germination and growth. The lectin is found not only in a freely soluble form but also in reversible association with particulate subcellular components. There appear to be no poly-peptides that can be solubilized with sonication and aqueous buffers from the embryo tissue that can interact with the agglutinin. This suggests that in vivo the lectin remains uncomplexed to endogenous glycoconjugates or is only able to bind to glycosylated integral membrane polypeptides. Alternatively the potential endogenous receptor(s) to wheat-germ agglutinin may not contain a polypeptide. Although the lectin is not present in the endosperm, seven polypeptides able to interact in a reversible way with wheat-germ agglutinin could be purified from that tissue.

Processing of a chloroplast-translated membrane protein in vivo. Analysis of the rapidly synthesized 32 000-dalton shield protein and its precursor in Spirodela oligorrhiza.

The 32 000-dalton (Da) shield protein regulating electron transport in Spirodela oligorrhiza is an integral chloroplast membrane polypeptide. It is rapidly synthesized, constituting a major chloroplast-translation product in vivo. Following in vitro translation of spirodela chloroplast RNA in a wheat germ system, a 33 500-Da polypeptide is produced. Synthesis of a 33 500-Da protein, associated with the chloroplast membrane, is also seen in vivo, within 2 min of pulse-labeling spirodela with radioactive amino acids. Comparative analyses among these polypeptides reveal: (a) all three are deficient in lysine residues; (b) the two 33 500-Da species have indistinguishable partial proteolytic digestion patterns while that for the 32 000-Da protein differs only slightly from them; (c) radioactivity from the 33 500-Da polypeptide is rapidly chased in vivo into the 32 000-Da protein, even in the presence of protein synthesis inhibitors. These results show the 33 500-Da proteins synthesized in vitro and in vivo to be the precursor form of the 32 000-Da shield protein in spirodela, with processing commencing only after completion of the precursor polypeptide chain and insertion into the membrane.

Characterization and localization of the transferrin receptor on rat retiulocytes

1. 1. A further characterization and localization of the membrane receptor for transferrin on rat reticulocytes is described. PAGE studies with a purified membrane complex B 2, from which the functional role in transferrin binding and iron uptake has been shown previously, showed that the transferrin receptor is localized on a membrane protein with a mol. wt of ~ 70–80.10 3. 2. 2. Selective solubilization of the rat reticulocyte membrane has shown that this receptor protein belongs to one of the minor integral membrane polypeptides, embedded in the lipid bilayer of the membrane. 3. 3. Proteolipid complexes, glycolipids and sialoglycoproteins of the rat reticulocyte membrane play no direct role in the binding capacity of the receptor.