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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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Mn-induced oligomerization of Golgi GPP130. (A) Immunoblots using anti-GPP130 antibodies to detect endogenous GPP130 after velocity gradient fractionation of detergent-solubilized cell lysates. Cells were either untreated (Ø) or treated with 0.5 mM MnCl2 for 3 h (Mn), followed by cross-linking with 0.5 mM DSP. The quantified distribution profile is also shown. The entirety of each fraction was analyzed, including the resuspended pellet (P). Gradients did not contain Mn. Results are representative of three trials. (B) Immunoblot and quantified profile using anti-GP73 to detect endogenous GP73 in an identical experiment. Results are representative of three trials. (C) Immunoblot to detect endogenous GPP130 in an identical experiment, except that the 3-h incubation (with or without Mn) was carried out at 20°C to arrest GPP130 in the TGN. Results are representative of three trials. (D) Immunoblot detection of transfected GP73 chimeric constructs containing GPP130 residues 36–175 without or with the 88AAAA91 substitution. Exactly as before, the cells were untreated or Mn treated, subjected to crosslinking, lysed, and fractionated on velocity gradients. The quantified fractionation profiles are also shown. Results are representative of three trials.
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Figure 4: Mn-induced oligomerization of Golgi GPP130. (A) Immunoblots using anti-GPP130 antibodies to detect endogenous GPP130 after velocity gradient fractionation of detergent-solubilized cell lysates. Cells were either untreated (Ø) or treated with 0.5 mM MnCl2 for 3 h (Mn), followed by cross-linking with 0.5 mM DSP. The quantified distribution profile is also shown. The entirety of each fraction was analyzed, including the resuspended pellet (P). Gradients did not contain Mn. Results are representative of three trials. (B) Immunoblot and quantified profile using anti-GP73 to detect endogenous GP73 in an identical experiment. Results are representative of three trials. (C) Immunoblot to detect endogenous GPP130 in an identical experiment, except that the 3-h incubation (with or without Mn) was carried out at 20°C to arrest GPP130 in the TGN. Results are representative of three trials. (D) Immunoblot detection of transfected GP73 chimeric constructs containing GPP130 residues 36–175 without or with the 88AAAA91 substitution. Exactly as before, the cells were untreated or Mn treated, subjected to crosslinking, lysed, and fractionated on velocity gradients. The quantified fractionation profiles are also shown. Results are representative of three trials.

Mentions: To test the hypothesis that Mn-induced oligomerization alters GPP130 trafficking, we first carried out cross-linking of untreated and Mn-treated cells using the membrane-permeant cross-linker dithiobis[succinimidyl propionate] (DSP). The Mn treatment was used to initiate GPP130 redistribution while leaving a substantial Golgi-localized pool. After cell lysis, the sedimentation behavior of endogenous GPP130 was determined on velocity gradients. In the absence of Mn, GPP130 was recovered near the top of the gradients, whereas Mn treatment resulted in recovery of almost 20% of the GPP130 in a much larger species near the bottom of the gradients (Figure 4A). Note that Mn was removed at the time of cross-linking and was absent for the remainder of the experiment, so the change in GPP130 behavior occurred in the intact cells. In addition, even in the absence of cross-linking, there was a reproducible, albeit small (5%), fraction of GPP130 in the bottom fractions of lysates from Mn-treated cells but not control cells (unpublished data). As a negative control, we also examined the behavior of endogenous GP73, which was recovered at the top of the gradients for both untreated and Mn-treated cells (Figure 4B). One concern was that the Mn-induced oligomerization of GPP130 might not be restricted to the Golgi. As a test, we carried out the cross-linking experiment on cells treated with Mn at 20°C to prevent GPP130 exit from the TGN. Immunofluorescence was used to confirm that 20°C incubation blocked redistribution of GPP130 into endosomes (unpublished data). Under these conditions, GPP130 was still recovered in the size-shifted fractions, indicating that oligomerization occurred in the Golgi (Figure 4C). Finally, we compared the oligomerization activity of the Mn-responsive wild-type GPP130 stem domain construct described earlier (Figure 1, residues 36–175 of GPP130 appended to the GP73 N-terminus) to the identically constructed protein containing the alanine substitutions that prevent Mn-induced trafficking to lysosomes (36-17588AAAA91). Whereas 20% of the 36–175 construct was recovered as a much larger species, the alanine-substituted construct was recovered exclusively in the top fractions of the gradients (Figure 4D).


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)

Mn-induced oligomerization of Golgi GPP130. (A) Immunoblots using anti-GPP130 antibodies to detect endogenous GPP130 after velocity gradient fractionation of detergent-solubilized cell lysates. Cells were either untreated (Ø) or treated with 0.5 mM MnCl2 for 3 h (Mn), followed by cross-linking with 0.5 mM DSP. The quantified distribution profile is also shown. The entirety of each fraction was analyzed, including the resuspended pellet (P). Gradients did not contain Mn. Results are representative of three trials. (B) Immunoblot and quantified profile using anti-GP73 to detect endogenous GP73 in an identical experiment. Results are representative of three trials. (C) Immunoblot to detect endogenous GPP130 in an identical experiment, except that the 3-h incubation (with or without Mn) was carried out at 20°C to arrest GPP130 in the TGN. Results are representative of three trials. (D) Immunoblot detection of transfected GP73 chimeric constructs containing GPP130 residues 36–175 without or with the 88AAAA91 substitution. Exactly as before, the cells were untreated or Mn treated, subjected to crosslinking, lysed, and fractionated on velocity gradients. The quantified fractionation profiles are also shown. Results are representative of three trials.
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Figure 4: Mn-induced oligomerization of Golgi GPP130. (A) Immunoblots using anti-GPP130 antibodies to detect endogenous GPP130 after velocity gradient fractionation of detergent-solubilized cell lysates. Cells were either untreated (Ø) or treated with 0.5 mM MnCl2 for 3 h (Mn), followed by cross-linking with 0.5 mM DSP. The quantified distribution profile is also shown. The entirety of each fraction was analyzed, including the resuspended pellet (P). Gradients did not contain Mn. Results are representative of three trials. (B) Immunoblot and quantified profile using anti-GP73 to detect endogenous GP73 in an identical experiment. Results are representative of three trials. (C) Immunoblot to detect endogenous GPP130 in an identical experiment, except that the 3-h incubation (with or without Mn) was carried out at 20°C to arrest GPP130 in the TGN. Results are representative of three trials. (D) Immunoblot detection of transfected GP73 chimeric constructs containing GPP130 residues 36–175 without or with the 88AAAA91 substitution. Exactly as before, the cells were untreated or Mn treated, subjected to crosslinking, lysed, and fractionated on velocity gradients. The quantified fractionation profiles are also shown. Results are representative of three trials.
Mentions: To test the hypothesis that Mn-induced oligomerization alters GPP130 trafficking, we first carried out cross-linking of untreated and Mn-treated cells using the membrane-permeant cross-linker dithiobis[succinimidyl propionate] (DSP). The Mn treatment was used to initiate GPP130 redistribution while leaving a substantial Golgi-localized pool. After cell lysis, the sedimentation behavior of endogenous GPP130 was determined on velocity gradients. In the absence of Mn, GPP130 was recovered near the top of the gradients, whereas Mn treatment resulted in recovery of almost 20% of the GPP130 in a much larger species near the bottom of the gradients (Figure 4A). Note that Mn was removed at the time of cross-linking and was absent for the remainder of the experiment, so the change in GPP130 behavior occurred in the intact cells. In addition, even in the absence of cross-linking, there was a reproducible, albeit small (5%), fraction of GPP130 in the bottom fractions of lysates from Mn-treated cells but not control cells (unpublished data). As a negative control, we also examined the behavior of endogenous GP73, which was recovered at the top of the gradients for both untreated and Mn-treated cells (Figure 4B). One concern was that the Mn-induced oligomerization of GPP130 might not be restricted to the Golgi. As a test, we carried out the cross-linking experiment on cells treated with Mn at 20°C to prevent GPP130 exit from the TGN. Immunofluorescence was used to confirm that 20°C incubation blocked redistribution of GPP130 into endosomes (unpublished data). Under these conditions, GPP130 was still recovered in the size-shifted fractions, indicating that oligomerization occurred in the Golgi (Figure 4C). Finally, we compared the oligomerization activity of the Mn-responsive wild-type GPP130 stem domain construct described earlier (Figure 1, residues 36–175 of GPP130 appended to the GP73 N-terminus) to the identically constructed protein containing the alanine substitutions that prevent Mn-induced trafficking to lysosomes (36-17588AAAA91). Whereas 20% of the 36–175 construct was recovered as a much larger species, the alanine-substituted construct was recovered exclusively in the top fractions of the gradients (Figure 4D).

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