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Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin.

Tewari R, Jarvela T, Linstedt AD - Mol. Biol. Cell (2014)

Bottom Line: Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes.Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn.These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

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Oligomerization of the purified lumenal stem domain. (A) Coomassie-stained gel and quantification indicating recovery of purified GST-tagged residues 36–247 after velocity gradient centrifugation in the absence or presence of 1 mM MnCl2. Result is representative of three independent trials. (B) Light scattering as indicated by absorbance at 350 nm at the indicated concentrations of MnCl2 for purified GST-tagged residues 36–247 of GPP130, GST-tagged GP73 stem domain, or GST alone (n = 5, ±SEM). (C) Light scattering after incubation at the indicated concentrations of MnCl2 for 10 min, followed by incubation for another 10 min with either no addition or addition of equimolar EDTA to chelate and reverse the effect of Mn binding (n = 3, ±SEM). (D) Light scattering for purified GST-tagged 36–175, 36–175 with the 88AAAA91 substitution, and GST alone (n = 3, ±SEM).
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Figure 3: Oligomerization of the purified lumenal stem domain. (A) Coomassie-stained gel and quantification indicating recovery of purified GST-tagged residues 36–247 after velocity gradient centrifugation in the absence or presence of 1 mM MnCl2. Result is representative of three independent trials. (B) Light scattering as indicated by absorbance at 350 nm at the indicated concentrations of MnCl2 for purified GST-tagged residues 36–247 of GPP130, GST-tagged GP73 stem domain, or GST alone (n = 5, ±SEM). (C) Light scattering after incubation at the indicated concentrations of MnCl2 for 10 min, followed by incubation for another 10 min with either no addition or addition of equimolar EDTA to chelate and reverse the effect of Mn binding (n = 3, ±SEM). (D) Light scattering for purified GST-tagged 36–175, 36–175 with the 88AAAA91 substitution, and GST alone (n = 3, ±SEM).

Mentions: Of interest, the purified GPP130 stem domain constructs 36–247 and 36–175 appeared to oligomerize in response to Mn addition. In the absence of added Mn, GST-GPP13036-247 was recovered roughly in the middle of velocity gradients, whereas addition of 1 mM MnCl2 shifted ∼60% of the protein toward the bottom of the gradients (Figure 3A). Mn-induced aggregation of the protein was also apparent simply by measuring absorbance at 350 nm, indicating light scattering (Cromwell et al., 2006). Whereas the two purified control proteins, GST and GST-tagged GP73 stem, failed to show an increase, the GST-GPP13036-247 protein yielded a robust increased absorbance clearly evident at 1 mM MnCl2 (Figure 3B). This effect was reversible because it was abrogated by a subsequent incubation with equimolar EDTA to chelate the Mn (Figure 3C). Of importance, the alanine substitutions that blocked GPP130 responsiveness in cells and reduced binding of Mn in the GST-GPP130 stem construct (36-17588AAAA91) also reduced the Mn-induced light scattering activity of this construct (Figure 3D). These results suggest that Mn binding might promote altered GPP130 trafficking by binding and inducing GPP130 oligomerization.


Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin.

Tewari R, Jarvela T, Linstedt AD - Mol. Biol. Cell (2014)

Oligomerization of the purified lumenal stem domain. (A) Coomassie-stained gel and quantification indicating recovery of purified GST-tagged residues 36–247 after velocity gradient centrifugation in the absence or presence of 1 mM MnCl2. Result is representative of three independent trials. (B) Light scattering as indicated by absorbance at 350 nm at the indicated concentrations of MnCl2 for purified GST-tagged residues 36–247 of GPP130, GST-tagged GP73 stem domain, or GST alone (n = 5, ±SEM). (C) Light scattering after incubation at the indicated concentrations of MnCl2 for 10 min, followed by incubation for another 10 min with either no addition or addition of equimolar EDTA to chelate and reverse the effect of Mn binding (n = 3, ±SEM). (D) Light scattering for purified GST-tagged 36–175, 36–175 with the 88AAAA91 substitution, and GST alone (n = 3, ±SEM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230593&req=5

Figure 3: Oligomerization of the purified lumenal stem domain. (A) Coomassie-stained gel and quantification indicating recovery of purified GST-tagged residues 36–247 after velocity gradient centrifugation in the absence or presence of 1 mM MnCl2. Result is representative of three independent trials. (B) Light scattering as indicated by absorbance at 350 nm at the indicated concentrations of MnCl2 for purified GST-tagged residues 36–247 of GPP130, GST-tagged GP73 stem domain, or GST alone (n = 5, ±SEM). (C) Light scattering after incubation at the indicated concentrations of MnCl2 for 10 min, followed by incubation for another 10 min with either no addition or addition of equimolar EDTA to chelate and reverse the effect of Mn binding (n = 3, ±SEM). (D) Light scattering for purified GST-tagged 36–175, 36–175 with the 88AAAA91 substitution, and GST alone (n = 3, ±SEM).
Mentions: Of interest, the purified GPP130 stem domain constructs 36–247 and 36–175 appeared to oligomerize in response to Mn addition. In the absence of added Mn, GST-GPP13036-247 was recovered roughly in the middle of velocity gradients, whereas addition of 1 mM MnCl2 shifted ∼60% of the protein toward the bottom of the gradients (Figure 3A). Mn-induced aggregation of the protein was also apparent simply by measuring absorbance at 350 nm, indicating light scattering (Cromwell et al., 2006). Whereas the two purified control proteins, GST and GST-tagged GP73 stem, failed to show an increase, the GST-GPP13036-247 protein yielded a robust increased absorbance clearly evident at 1 mM MnCl2 (Figure 3B). This effect was reversible because it was abrogated by a subsequent incubation with equimolar EDTA to chelate the Mn (Figure 3C). Of importance, the alanine substitutions that blocked GPP130 responsiveness in cells and reduced binding of Mn in the GST-GPP130 stem construct (36-17588AAAA91) also reduced the Mn-induced light scattering activity of this construct (Figure 3D). These results suggest that Mn binding might promote altered GPP130 trafficking by binding and inducing GPP130 oligomerization.

Bottom Line: Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes.Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn.These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

Show MeSH
Related in: MedlinePlus