Urea Reduces the Aggregation of Membrane Proteins on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Soulié, Stéphanie; Møller, Jesper; Falson, Pierre; Maire, Marc le

doi:10.1006/abio.1996.0183

Cited by 35 publications

(25 citation statements)

References 2 publications

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“…Other signaling molecules that form SDS-resistant oligomers include: enzymes such as Na + /K + -ATPase and eNOS; the GPCR, rhodopsin (Soulie et al, 1996); and members of the AKAP family, especially AKAP12. AKAP12 requires 8M urea for dissociation of oligomers (Tao et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes

Yokoyama

Golebiewska

Wang

et al. 2010

Journal of Cell Science

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SummaryDishevelled-3 (Dvl3) is a multivalent scaffold protein that is essential to Wnt signaling during development. Although Dvl-based punctae have been visualized by fluorescence microscopy; the physical nature and dynamic character of the such complexes are enigmatic. We use steric-exclusion chromatography, affinity pull-downs, proteomics and fluorescence correlation microscopy to characterize supermolecular Dvl3-based complexes of totipotent mouse F9 cells. The molecular mass of the complexes ranges from that of homodimeric Dvl3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa. Addition of Wnt3a stimulates the formation of Dvl3-based complexes of greater molecular mass within 30 minutes. The presence of DKK1 and knockdown of Dishevelled proteins block formation of the 2 MDa Dvl3-based complexes and also block Wnt3a stimulation of the canonical pathway. Fluorescent correlation microscopy identified supermolecular Dvl3-based complexes with a molecular mass >30 MDa in live cells; these complexes were provoked to form structures with even greater molecular mass by Wnt3a. We establish for the first time the physical and functional nature of very large, supermolecular Dvl3-based complexes.

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Section: Discussionmentioning

confidence: 99%

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes

Yokoyama

Golebiewska

Wang

et al. 2010

Journal of Cell Science

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“…The elution of this material by gel filtration, in the presence of SDS, in fractions predicted to contain molecules in excess of 600 kDa and the appearance of this material on SDS-PAGE as a smear ranging from 65 kDa to the top of the gel suggest an unusual physical state. However, multispanning membrane proteins can undergo aggregation under denaturing conditions (40,45). We therefore checked to see whether the appearance by SDS-PAGE of the galactose oxidase-NaB[…”

Section: Discussionmentioning

confidence: 99%

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

et al. 2009

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A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB 3 H 4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecularweight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB 3 H 4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

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“…containing 7 M urea, confirming they are not formed on the SDS-polyacrylamide gels and suggesting they were associated with complexes of high molecular masses. Urea is known to suppress non-specific aggregation of membrane proteins in SDS-polyacrylamide gels (Soulié et al, 1996). On the blot of the gel probed with mAb to colicin A, colicins Au were detected without monomeric colicin A in the unheated samples of the insoluble fractions treated with urea.…”

Section: Pools Of Released and Unreleased Colicin Amentioning

confidence: 99%

Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A–porin complexes

Cavard

2004

Microbiology

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Release of colicin A was studied in Escherichia coli cells that differed in expressing the colicin A lysis protein (Cal). Pools of released and unreleased colicin A were harvested throughout colicin A induction. The amount of colicin A in each pool varied with the time of induction, allowing the definition of two sequential steps in colicin A release, one of which was dependent on Cal. Each step of colicin A release was differently affected in cells containing Cal mutants in which the N-terminal cysteine residue was substituted by either proline or threonine, preventing them from being acylated and matured. These Cal mutants were only observed in degP cells, indicating that the DegP protease cleaved the unacylated precursor of Cal. Cal was found in the insoluble fraction of the pools of released and unreleased colicin A together with the hetero-oligomers of colicin A and porins (colicins Au). The biogenesis of colicins Au was studied in temperature-sensitive secA and secY strains and found to be Sec-independent, indicating that they are formed by newly synthesized colicin A binding to mature porins already incorporated in the outer membrane. Cal is a lipoprotein similar to VirB7, a constituent of the type IV secretion system. It would interact with colicins Au to constitute the colicin A export machinery.

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Urea Reduces the Aggregation of Membrane Proteins on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Cited by 35 publications

References 2 publications

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A–porin complexes

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