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F G van der Goot

Publications and source records attributed to F G van der Goot.

At least 19 recordsLinked to original sources
PubMed2002

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.

Aeromonas hydrophila↗
PubMed2001

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Animals↗
PubMed2001

Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack.

While the existence of cholesterol/sphingolipid (raft) membrane domains in the plasma membrane is now supported by strong experimental evidence, the structure of these domains, their size, their dynamics, and their molecular composition remain to be understood. Raft domains are thought to represent a specific physical state of lipid bilayers, the liquid-ordered phase. Recent observations suggest that in the mammalian plasma membrane small raft domains in ordered lipid phases are in a dynamic equilibrium with a less ordered membrane environment. Rafts may be enlarged and/or stabilized by protein-mediated cross-linking of raft-associated components. These changes of plasma membrane structure are perceived by the cells as signals, most likely an important element of immunoreceptor signalling. Pathogens abuse raft domains on the host cell plasma membrane as concentration devices, as signalling platforms and/or entry sites into the cell. Elucidation of these interactions requires a detailed understanding raft structure and dynamics.

Animals↗
PubMed2001

Not as simple as just punching a hole.

Like a variety of other pathogenic bacteria, Aeromonas hydrophila secretes a pore-forming toxin that contribute to its virulence. The last decade has not only increased our knowledge about the structure of this toxin, called aerolysin, but has also shed light on how it interacts with its target cell and how the cell reacts to this stress. Whereas pore-forming toxins are generally thought to lead to brutal death by osmotic lysis of the cell, based on what is observed for erythrocytes, recent studies have started to reveal far more complicated pathways leading to death of nucleated mammalian cells.

Aeromonas↗
PubMed2001

The bacterial toxin toolkit.

Pathogenic bacteria and higher eukaryotes have spent a long time together, leading to a precise understanding of one another's way of functioning. Through rapid evolution, bacteria have engineered increasingly sophisticated weapons to hit exactly where it hurts, interfering with fundamental host functions. However, toxins are not only useful to the bacteria - they have also become an essential asset for life scientists, who can now use them as toolkits to explore cellular processes.

Bacterial Toxins↗
PubMed2000

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis.

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.

Amino Acid Sequence↗
PubMed2000

Adventures of a pore-forming toxin at the target cell surface.

The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.

Aeromonas hydrophila↗
PubMed2000

Surface dynamics of aerolysin on the plasma membrane of living cells.

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Animals↗
PubMed1999

Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding.

The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.

Animals↗
PubMed1999

Increased stability upon heptamerization of the pore-forming toxin aerolysin.

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Amino Acid Sequence↗
PubMed1999

Membrane interaction of TNF is not sufficient to trigger increase in membrane conductance in mammalian cells.

Tumor necrosis factor TNF can trigger increases in membrane conductance of mammalian cells in a receptor-independent manner via its lectin-like domain. A lectin-deficient TNF mutant, lacking this activity, was able to bind to artificial liposomes in a pH-dependent manner, but not to insert into the bilayer, just like wild type TNF. A peptide mimicking the lectin-like domain, which can still trigger increases in membrane currents in cells, failed to interact with liposomes. Thus, the capacity of TNF to trigger increases in membrane conductance in mammalian cells does not correlate with its ability to interact with membranes, suggesting that the cytokine does not form channels itself, but rather interacts with endogenous ion channels or with plasma membrane proteins that are coupled to ion channels.

Amino Acid Sequence↗
PubMed1999

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.

Binding Sites↗
PubMed1999

The staphylococcal alpha-toxin pore has a flexible conformation.

The alpha-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the alpha-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted alpha-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane beta-barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the alpha-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation.

Bacterial Toxins↗
PubMed1999

The lectin-like domain of tumor necrosis factor-alpha increases membrane conductance in microvascular endothelial cells and peritoneal macrophages.

Herein, we show that TNF exerts a pH-dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor-independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin-like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17-amino acid peptide of TNF, which was previously shown to exert lectin-like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenous ion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology.

Animals↗