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S H Keller

Publications and source records attributed to S H Keller.

11 recordsLinked to original sources
PubMed2001

Adjacent basic amino acid residues recognized by the COP I complex and ubiquitination govern endoplasmic reticulum to cell surface trafficking of the nicotinic acetylcholine receptor alpha-Subunit.

The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of alpha-gamma-alpha-delta-beta. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg(313)-Lys(314) sequence in the large cytoplasmic loop of the alpha-subunit to K314Q promotes the trafficking of the mutant unassembled alpha-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q alpha-subunit with gamma-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type alpha-subunits when co-expressed with the beta-, delta-, and gamma-subunits. This suggests that the Arg(313)-Lys(314) sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the alpha-subunit. Although unassembled K314Q alpha-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q alpha-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface.

Animals↗
PubMed1998

Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor.

To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway.

Acetylcysteine↗
PubMed1996

Involvement of the chaperone protein calnexin and the acetylcholine receptor beta-subunit in the assembly and cell surface expression of the receptor.

The nicotinic acetylcholine receptor at the neuromuscular junction is a ligand-gated ion channel assembled in the endoplasmic reticulum from four distinct glycoprotein subunits into the pentameric configuration of alpha2betagammadelta. The individual homologous subunits form specific contacts at interfaces with neighboring subunits to achieve the appropriate orientation and order of each subunit in surrounding the ion channel. Assembly is thought to proceed through the formation of intermediates composed of dimers of the alphadelta and alphagamma subunits which are eventually joined by the beta-subunit to achieve a circular structure enclosing the gated ion channel. In this study, we transfect cDNAs encoding receptor subunits in various combinations into HEK-293 cells to identify intracellular factors that influence the assembly and cell surface expression of the receptor. Our data derived from brefeldin A-treated cells indicate that intracellular association of the receptor subunits with the beta-subunit increases the pool of fully assembled receptors available for transport to the cell surface, presumably by protection from degradation. In addition, we determined that the chaperone protein calnexin is associated with the isolated alpha-, beta-, and delta-subunits of the receptor, but calnexin is not detected in association with assembled alphadelta subunit dimers. Calnexin is also detected in association with maturely folded, unassembled alpha-subunits, as observed by the recognition of this complex by the monoclonal antibody mAb 35, believed to be specific for correctly folded alpha-subunits. Thus, calnexin appears to associate with the individual nascent subunits, thereby facilitating their assembly into the mature pentameric receptor.

Animals↗
PubMed1995

Molecular determinants conferring alpha-toxin resistance in recombinant DNA-derived acetylcholine receptors.

Sequences of the alpha-subunits of the nicotinic acetylcholine receptor from the snake and mongoose contain several differences in the region between amino acids 183 and 200. Receptors from both of these species reveal resistance to the snake alpha-toxins presumably arising as a protective evolutionary mechanism. Sequence differences include the added glycosylation signals at residue 187 in the mongoose and at residues 189 and 111 in snake. Although previous observations with peptides and fusion proteins either synthesized chemically or in a bacterial expression system indicate that certain amino acid residues may contribute to the resistance, our findings with the intact receptor in an eukaryotic expression system indicate the major role for glycosylation. In this study, we show that addition of glycosylation signals gives rise to virtually complete glycosylation at the added sites, although heterogeneity of oligosaccharide processing is evident. By analysis of combinations of mutants, we document that glycosylation exerts the predominant influence on alpha-toxin binding. Substitutions at other residues are largely without influence as single mutations but appear to decrease affinity further in multiple mutants, particularly where the receptor is glycosylated at the 187 and 189 positions. Glycosylation exerts a major influence on the dissociation as well as the association rates of the alpha-toxin-receptor complex, suggesting that the decrease for alpha-toxin affinity is not simply a consequence of restricted diffusional access, rather glycosylation affects the conformation and stability of the bound complex.

Amino Acid Sequence↗
PubMed1994

The isolation of acrosome-reaction-inducing glycoproteins from sea urchin egg jelly.

The solubilized jelly coat of Strongylocentrotus purpuratus eggs was boiled in SDS and mercaptoethanol, precipitated in ethanol, washed in 70% ethanol, lyophilized, and redissolved in seawater. The sample retained its activity to induce the sperm acrosome reaction (AR), showing that the AR inducer was resistant to harsh denaturing conditions. Egg jelly (EJ) was separated into its component macromolecules by denaturing polyacrylamide gel electrophoresis and the gel silver-stained. Several macromolecules ranging from approximately 30 to 380 kDa were consistently observed. These macromolecules were not seen by Coomassie blue staining. With the exception of the fucose sulfate polymer (FSP; 380 kDa) and the 300-kDa component, the other molecules were of minor abundance. Sephacryl-500 gel-filtration chromatography in detergents and disulfide reducing agents separated EJ into three fractions. The FSP and 300-kDa components were purified to homogeneity. A third fraction consisted of components of 30, 82, 116, and 138 kDa. The purified FSP and 300-kDa components had insignificant AR-inducing activity. Substantial AR activity was found only in the 30 to 138-kDa fraction. Ion-exchange chromatography and Centricon filtration separated the 82- and 138-kDa glycoproteins from the other components, but these two components could not be separated from each other. There may be other AR-inducing molecules in sea urchin EJ that were not visible after silver staining of gels. Also, some EJ components are so large that they did not enter the stacking gel. Of those macromolecules that were visible on gels after silver staining, only the fraction containing the 80- and 138-kDa proteins had significant AR-inducing activity.

Acrosome↗
PubMed1994

The sea urchin egg jelly coat consists of globular glycoproteins bound to a fibrous fucan superstructure.

Intact egg jelly (EJ) coats surrounding eggs of the sea urchin Strongylocentrotus purpuratus were visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, rotary-shadowing technique. The hydrated EJ coat forms an extensive fibrous network that makes contact with the vitelline layer at the egg surface. Fibers are decorated along their length with particles, particle density being highest in the interior regions of the coat. The macromolecular components making up the EJ network were visualized by rotary-shadowing of mica-adsorbed EJ samples. Whole EJ coats solubilized in pH 5 sea-water and spread on the mica surface consist of complex networks of branching fibers decorated with large patches of amorphous material. As we have previously shown (Keller and Vacquier, 1994), EJ boiled in a dissolution buffer containing SDS and beta-mercaptoethanol and applied to a Sephacryl-500 gel filtration column can be separated into three fractions: a 380-kDa fucose sulfate polymer (FSP), which elutes in the void volume, and two column-included fractions consisting of intermediate (300 kDa) and low-molecular-weight (30- to 138-kDa) glycoproteins. Rotary-shadowing of the FSP fraction reveals branched fibrous components similar in appearance to that of solubilized whole EJ but devoid of any particulate decoration. In contrast, intermediate- and low-molecular-weight EJ components are strictly globular in appearance but are distinguishable on the basis of size. Ion-exchange purification of whole EJ yields two glycoproteins, of 82 and 138 kDa, having AR-inducing activity (Keller and Vacquier, 1994). Platinum replication shows these active components to be small spherical molecules about 8 nm in diameter. The above fractionation scheme requires harsh dissociation conditions. Indeed, if EJ is not boiled in SDS buffer before fractionation, the 300-kDa fraction and the FSP appear together in the void volume. Rotary-shadowing of this complex reveals a multistranded polymer, decorated with glycoproteins at specific kink points. Taken together, our data suggest that the EJ network is composed of a fucose sulfate polymer superstructure to which glycoproteins are bound.

Animals↗
PubMed1989

Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata.

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.

1-Methyl-3-isobutylxanthine↗