Dissociation of the Octameric Enolase from S. Pyogenes - One Interface Stabilizes Another

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO4, a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO4 dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable

CD spectra of the variant enolase in its octameric and monomeric forms.

<p>The concentration of enolase in all samples was 4.1 µM. Samples were incubated for 18 hours at 20°C; the CD spectra were then recorded as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#s4" target="_blank">Methods</a>. Solid line, enzyme in TME; dotted line, enzyme in TME plus 0.2 M NaClO₄; dashed line, enzyme in glycine buffer, pH 10.</p

Dissociation and inactivation of the wild type octameric enolase and the F137L/E363G variant by NaClO₄.

<p>Enolase, 2.7 µM was incubated in NaClO₄ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g003" target="_blank">3</a>. Prior to loading the AUC cells, each sample was assayed for enzymatic activity. Open symbols, % activity; closed symbols, % non-monomeric protein (calculated at 100% - % monomeric); circles, F137L/E363G variant; triangles, wild type enolase.</p

Sedimentation velocity data for the dissociation of the octameric enolase by NaClO₄.

<p>The concentrations of the various species in the sample are shown as a function of their sedimentation coefficients. Prior to centrifugation, the samples were incubated for 18 hours at 15°C in TME (solid line), TME containing 0.2 M NaClO₄ (long dashes) or TME containing 0.28 M NaClO₄ (dash-dot-dot). All samples contained 2.7 µM enolase. Centrifugation and data analysis were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#s4" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g002" target="_blank">Fig. 2B</a> is an enlargement of the data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g002" target="_blank">Fig. 2A</a>.</p

Distribution of species versus concentration of NaClO₄.

<p>Data were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g002" target="_blank">Fig. 2</a>. Data for each concentration of NaClO₄ came from separate incubations and AUC runs, performed over a period of several days. All samples contained 2.7 µM enolase. Solid circles, % octameric; open circles, % monomeric; solid triangles, % all intermediate species.</p

The structure of the octameric enolase of <i>S. pneumoniae</i> (1W6T.pdb).

<p>A) The dimeric unit showing the monomer-monomer interface, viewed down the β-barrel of the large domain of one subunit. The green ball is the Mg²⁺ at the active site. B) The octamer, with one dimer colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g001" target="_blank">Fig. 1A. C</a>) Close up of the dimer-dimer interface, showing F137 (bottom of the figure) and E362 (top pair of residues); residue 362 in <i>S. pneumoniae</i> = residue 363 in <i>S. pyogenes</i>. One dimer is blue and the other is green, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g001" target="_blank">Fig. 1B</a>. All figures were made with Chimera (<a href="http://www.cgl.ucsf.edu/chimera/" target="_blank">www.cgl.ucsf.edu/chimera/</a>).</p

Distribution of species versus concentration of NaClO₄ for the F137L/E363G variant.

<p>Data were obtained from experiments performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008810#pone-0008810-g003" target="_blank">3</a>. All samples contained 2.7 µM enolase. Solid circles, % octameric; open circles, % monomeric; solid triangles, % all intermediate species.</p

Time course of changes in DLS intensity and activity during incubation in NaClO₄.

<p>NaClO₄ was added to a solution of 2.7 µM enolase; the sample was immediately placed in the DLS cuvette and measurements begun or aliquots were taken at various times for activity measurements. For both DLS and activity measurements, the sample was held at 15°C. Open circles, DLS intensity; solid circles, activity.</p

CED-10/Rac1 Regulates Endocytic Recycling through the RAB-5 GAP TBC-2

<div><p>Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the <em>Caenorhabditis elegans</em> intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in <em>ced-12</em>, <em>ced-10</em>, and <em>tbc-2</em> mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.</p> </div

Expression of GTPase-defective RAB-5 interferes with the trafficking of recycling cargo.

<p>(A and B) Confocal images of recycling cargo hTAC-GFP in the intestinal epithelium. Wild-type animals (A) and animals expressing of GTPase-defective RAB-5 (tagRFP-RAB-5(Q78L)) (B) are shown. (C) Quantification of hTAC-GFP puncta and tubule intensity. (D and E) Confocal images of recycling cargo hTfR-GFP in the intestinal epithelium. Wild-type animals (D) and animals expressing of GTPase-defective RAB-5 (tagRFP-RAB-5(Q78L)) (E) are shown. (F) Quantification of hTfR-GFP puncta intensity. Error bars represent standard deviations from the mean (n = 18 each, 6 animals of each genotype sampled in three different regions of each intestine). Asterisks indicate a significant difference in the one-tailed Student's T-test (***p<0.0001). Scale bar, 10 µm.</p