Removal Of Peripheral Proteins Research Articles

Ribosome-independent Regulation of Translocon Composition and Sec61α 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.

Characterization of structural and functional phosphoinositide domains in human erythrocyte membranes.

In the erythrocyte membrane, only a fraction (50-60%) of phosphatidylinositol 4,5-bisphosphate (PIP2) and of phosphatidylinositol 4-phosphate (PIP) is rapidly turned over by specific kinases and phosphatases and accessible to hydrolysis by the polyphosphoinositide (PPI)-specific phospholipase C (PLC). To investigate whether the metabolic segregation of PPI resulted from preferential interactions with proteins, we have measured the accessibility of PPI to bee venom phospholipase A2 (PLA2) in native erythrocyte membranes, or after treatments designed to remove peripheral proteins and cytoplasmic domains of integral proteins. In native membranes, PPI, as well as the other major phospholipids, behaved as two distinct fractions (R1 and R2) differing by their sensitivity to PLA2. Such a behavior was not observed in PIP and PIP2 containing artificial vesicles. Evidence was provided that the highly sensitive fraction of PIP and PIP2 (R1) may be identical to the PLC-sensitive and rapidly metabolized pool. Removal of peripheral proteins, followed by proteolysis of the cytoplasmic domain of integral proteins, mainly glycophorins and band 3, led to a reduction of the R1 fraction of PIP and of PIP2. It is proposed that the rapidly metabolized pool of PIP2 and PIP, involved in the regulation of major cellular functions, would be maintained in its functional state through interactions with integral proteins.

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The “apparent soluble” form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.

Characterization of Mg 2+-ATPase from slow-twitch muscle membranes

1. 1. SR vesicles from rabbit slow-twitch muscle reveal high activity (0.7–0.9 μmol/mg × min) of “basic” or Mg 2+-ATPase. 2. 2. This enzyme differs in its biochemical properties from the well characterized Ca 2+ pump ATPase. 3. 3. It is active in millimolar concentration of magnesium or calcium. The activity is inhibited by various detergents except for digitonin. 4. 4. This enzyme seems to be an integral membrane protein since it remains in the membrane after removal of peripheral proteins with EDTA. 5. 5. It can be partially solubilized from the membrane using digitonin without a decrease in specific activity. 6. 6. Ion exchange chromatography on DEAE-Sephacel of the post digitonin supernatant allows us to obtain a 5-fold increase in Mg 2+-ATPase specific activity concomitantly with the enrichment in two proteins of M r = 30, 000 and 150, 000.

The preparation of hydrophilic derivatives of band 3 protein by acylation of the human red blood cell membrane.

In situ reaction of erythrocyte membranes with dicarboxylic anhydrides leads to solubilization of hydrophobic integral proteins. Removal of peripheral proteins and bulk lipid by appropriate sedimentation and dialysis steps yields hydrophilic band 3 protein derivatives. These acyl compounds display size heterogeneity upon gel filtration. A chromatographically homogeneous acyl band 3 protein is obtained if the acylation is conducted in the presence of detergent and the detergent subsequently removed. Hydrophilic acyl derivatives of band 3 protein can be subjected to conventional analytical techniques without the use of detergents.