Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High-and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.
Wild type and mutant phosphate carriers (PIC) fromSaccharomyces cerevisiae mitochondria were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and optimally reconstituted into liposomal membranes. This PIC can function as coupled antiport (P i ؊ / P i ؊ antiport and P i ؊ net transport, i.e. P i ؊ /OH ؊ antiport) and uncoupled uniport (mercuric chloride-induced P i ؊ efflux). The basic kinetic properties of these three transport modes were analyzed. The kinetic properties closely resemble those of the reconstituted PIC from beef heart mitochondria. A competitive inhibitor of phosphate transport by the PIC, phosphonoformic acid, was used to establish functional overlap between the the physiological transport modes and the induced efflux mode. Replacement mutants were used to relate the reversible switch from antiport to uniport to a specific residue of the carrier. There are only three cysteines in the yeast PIC. They are at positions 28, 134, and 300 and were replaced by serine, both individually and in combinations. Cysteine 300 near the C-terminal loop and cysteine 134 located within the third transmembrane segment are accessible to bulky hydrophilic reagents from the cytosolic side, whereas cysteine 28 within the first transmembrane segment is not. None of the three cysteines is relevant to the two antiport modes. Cysteine 134 was identified to be the major target of bulky SH reagents, that lead to complete inactivation of the physiological transport modes. The reversible conversion between coupled antiport and uncoupled uniport of the PIC depends on the presence of one single cysteine (cysteine 28) in the PIC monomer, i.e. two cysteines in the functionally active dimer. The consequences of this result with respect to a functional model of the carrier protein are discussed.
Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C 12 E 8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo-and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.
Thirty-two genes have been identified within the genome of the yeast Saccharomyces cerevisiae which putatively encode mitochondrial transport proteins. We have attempted to overexpress a subset of these genes, namely those which encode mitochondrial transporters of unknown function, and have succeeded in overexpressing 19 of these genes. The overexpressed proteins were then isolated and tested for five well-characterized reconstituted transport activities (i.e., the transport of citrate, dicarboxylates, pyruvate, camitine, and aspartate). Utilizing this approach, we have clearly identified the yeast mitochondrial dicarboxylate transport protein, as well as two additional lower-magnitude transport functions (i.e., tricarboxylate and dicarboxylate transport activities). The implications of these results and the considerations relevant to this approach are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.