Digitonin-solubilized Membranes Research Articles

Svp26 facilitates ER exit of mannosyltransferases Mnt2 and Mnt3 in Saccharomyces cerevisiae.

After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.

PEX14 is required for microtubule-based peroxisome motility in human cells

We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.

Characterization of Rat TOM40, a Central Component of the Preprotein Translocase of the Mitochondrial Outer Membrane

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable alpha-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a beta-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitonin-solubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a approximately 400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.

Identification and characterization of guanine nucleotide-sensitive melatonin receptors in chicken brain

Specific melatonin receptors present in crude membrane preparations of chicken forebrain were identified by specific binding activity of 2-[ 125I]iodomelatonin and melatonin-dependent inhibition of forskolin-stimulated adenylate cyclase activity by a pertussis toxin-sensitive mechanism. The membrane-bound melatonin receptors were successfully solubilized with digitonin, and the ligand binding activity was shown to be preserved in solubilized membranes when compared with that of crude membranes. In both fractions, melatonin receptors consisted of two binding components with picomolar and nanomolar affinity. The ligand binding activity of solubilized receptors was decreased after treatment with guanine nucleotides, and guanosine 5 - ́ (γ-thio)triphosphate (GTPγS) induced a transformation of the high affinity receptors to low affinity state. Moreover, [ 35S]GTPγS was indicated to bind to solubilized membranes in a melatonin-dependent fashion. These results demonstrate the existence of functional melatonin receptors-G proteins coupling in digitonin-solubilized membranes of chicken forebrain membranes.

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.

Recognition by anti-idiotypic anti-thyrotropin-releasing hormone (TRH) antibody of rat TRH receptors

We asked whether anti-idiotypic antibodies raised against anti-TRH antibody could bind to TRH receptors in the rat anterior pituitary and brain. Six rabbits were immunized with IgG from a rabbit anti-TRH antiserum. One anti-idiotypic antibody caused strong, dose-dependent inhibition in anti-TRH antibody-binding to [ 125]I-TRH. This inhibition was not observed after treatment with goat anti-rabbit IgG antibody. The anti-idiotypic anti-TRH antibody significantly immunoprecipitated digitonin-solubilized pituitary TRH receptors. When eluates of digitonin-solubilized membranes which were adsorbed by either an anti-idiotypic anti-TRH IgG-, normal rabbit IgG-linked affinity column or control column were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by silver stain, only the former eluate showed two bands under nonreducing conditions; one corresponded to a molecular weight marker of 200K, the other to 100K. Western blotting analysis with an anti-idiotypic anti-TRH antiserum showed a single band of molecular weight 56K under reducing conditions. The present study indicates that one can make anti-idiotypic antibodies that specifically recognize TRH receptors.

Purification and characterization of the rat pancreatic cholecystokinin receptor

Cholecystokinin (CCK) is a peptide hormone that has a variety of physiologically important functions in the gastrointestinal tract, in which distinct high affinity receptors have been identified. We describe here the purification of the digitonin-solubilized rat pancreatic receptor as an initial step in the determination of its primary structure. Solubilization of total pancreatic membranes using 1% digitonin resulted in a single class of binding sites with a specific content of 4 pmol/mg as measured in a soluble binding assay using the nonpeptidyl CCK antagonist [3H]3S[-]-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl]-1H-indole-2-carboxamide [( 3H]364,718). The solubilized receptor was purified using the following chromatographic steps: 1) cation exchange; 2) Ulex europaeus agglutinin-I-agarose; and 3) Sephacryl S-300. The final preparation of the purified receptor had a specific content of 8,055 pmol/mg, which represented a 9,051-fold purification from intact membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified receptor preparation under reducing conditions resulted in a predominant polypeptide with an Mr = 85,000-95,000 and minor polypeptides of Mr = 57,000 and 26,000 as determined by radiolabeling and silver staining. Solubilized pancreatic membranes were affinity labeled with the peptidyl CCK agonist 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Phe33)CCK-26-33] and chromatographed under conditions similar to those described for untreated membranes. Elution of radioactive peaks from each chromatographic column was coincident with [3H]364,718 binding activity and resulted in a labeled polypeptide having the same electrophoretic mobility as receptor derived from freshly labeled membranes and purified from untreated membranes. High performance liquid-gel exclusion chromatography of the crude digitonin-solubilized membrane preparation revealed an estimated molecular size for the [3H]364,718-binding activity of 370,000, which was consistent with the size determined by nondenaturing gel electrophoresis of the purified receptor complexed with the labeled nonpeptidyl antagonist. Binding of [3H]364,718 to the purified receptor preparation was comparable to that observed with the crude solubilized pancreatic membrane preparation; and both the homologous ligand 364,718 (Ki = 0.5 nm) and CCK-8 (Ki = 1.4 microM) competed for binding to both preparations in a similar manner.

Characterization and solubilization of cyclo(His-Pro) binding from rat liver membranes.

We have found cyclo(His-Pro) binding in rat liver plasma membranes. This study focused on the characterization of solubilized binding for cyclo(His-Pro) in rat liver membranes. The cyclo(His-Pro) binding of liver membranes was solubilized by digitonin and octyl-glucopyranoside. The efficiency of solubilization with digitonin was greater. However, cyclo(His-Pro) binding was not solubilized by Triton X-100, CHAPS, or Lubrol. Digitonin-solubilized membranes showed cyclo(His-Pro) binding with a high affinity constant (17 nM) and a low binding capacity (38 fmol/mg protein). Lectins from wheat germ, Bandeiraea simplicifolia II, Dolichos biflorus, Glycine max, and Tetragonolobus purpureas significantly adsorbed [3H]cyclo(His-Pro)-binding complex, but Bandeiraea simplicifolia I, Ricinus communis I, or Lens culinaris did not adsorb the binding complex. An analysis of [3H]cyclo(His-Pro)-associated membranes by high performance gel filtration chromatography showed a radioactive peak of Mr 200,000. These data indicate that cyclo(His-Pro) binding of rat liver membranes is solubilized by digitonin and is a glycoprotein of Mr 200,000.

3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland

AHN 086, an isothiocyanato derivative of Ro 5-4864 (4′-chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [ 3H]AHN 086 labels a ≈ 30 kDa protein in the rat pineal gland determined by both SDS-polyacrylamide gel electrophoresis and gel filtration high-performance liquid chromatography of digitonin-solubilized membranes. Specific incorporation of [ 3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [ 3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high-affinity binding sites for Ro 5-4864) or ovalbumin. These findings suggest that the ≈ kDa protein labeled by [ 3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.

Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases

We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:beta-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1-->3)-beta-glucan synthase on both Ca(2+) and a beta-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.

Solubilization of the platelet plasma membrane serotonin transporter in an active form.

The activity of the serotonin transporter of platelet plasma membrane may be assessed by its ability to bind [N-methyl-3H]imipramine, even in the presence of detergents which disrupt the membrane and inactivate serotonin transport. Of the 21 detergents which were tested for their ability to solubilize the transporter in an active form, some, such as Triton X-100, inactivate imipramine-binding activity in a manner which is difficult or impossible to reverse, while inactivation by others, such as cholate, is readily reversed by gel filtration. Of the detergents which do not inactivate imipramine binding, most, such as the Tween series, also fail to solubilize significant amounts of the transporter. Of all the detergents tested, only one, digitonin, solubilized specific imipramine-binding activity. Imipramine binds to the digitonin-solubilized transporter with slightly reduced affinity, but in all other respects binding to intact and digitonin-solubilized membranes is identical. Thus, binding is, in both cases, saturable and Na+-dependent, and is reversed by serotonin. Furthermore, inhibitors of serotonin transport but not of serotonin-mediated platelet aggregation block imipramine binding to the soluble preparation. Sodium-dependent binding to the soluble extract is insensitive to K+, suggesting that separate sites on the serotonin transporter mediate the effects of effects of Na+ and K+.