Manganese-induced trafficking and turnover of the cis-Golgi glycoprotein GPP130

Abstract

Manganese is an essential element that is also neurotoxic at elevated exposure. However, mechanisms regulating Mn homeostasis in mammalian cells are largely unknown. Because increases in cytosolic Mn induce rapid changes in the localization of proteins involved in regulating intracellular Mn concentrations in yeast, we were intrigued to discover that low concentrations of extracellular Mn induced rapid redistribution of the mammalian cis-Golgi glycoprotein Golgi phosphoprotein of 130 kDa (GPP130) to multivesicular bodies. GPP130 was subsequently degraded in lysosomes. The Mn-induced trafficking of GPP130 occurred from the Golgi via a Rab-7-dependent pathway and did not require its transit through the plasma membrane or early endosomes. Although the cytoplasmic domain of GPP130 was dispensable for its ability to respond to Mn, its lumenal stem domain was required and it had to be targeted to the cis-Golgi for the Mn response to occur. Remarkably, the stem domain was sufficient to confer Mn sensitivity to another cis-Golgi protein. Our results identify the stem domain of GPP130 as a novel Mn sensor in the Golgi lumen of mammalian cells.

Figure 1.

Mn induces the degradation of GPP130 in HeLa cells. (A) HeLa cells were treated with 500 μM MnCl₂ for the times indicated and subjected to immunoblot analyses to detect GPP130, GP73, and α-tubulin. (B) Levels of GPP130 and GP73 were quantified at the 0- and 8-h time points and normalized to tubulin levels (mean ± SE, n = 3; p < 0.05). (C) HeLa cells on coverslips were untreated or treated with 500 μM MnCl₂ for 8 h, fixed, and costained using anti-GPP130 (using the anti-GPP130 mAb) and anti-GP73 antibodies. Bar, 10 μm.

Figure 2.

GPP130 redistributes to MVBs and lysosomes in response to Mn. (A) HeLa cells were treated with 500 μM MnCl₂ for the times indicated, fixed, and costained to detect GPP130 and giantin. Bar, 10 μm. (B and C) HeLa cells were transfected with GFP-tagged Rab7 WT, and 24 h after transfection they were treated with 500 μM MnCl₂ for 2 h and stained to detect GPP130. Arrowheads indicate overlap between GPP130 and Rab7-GFP. Bar, 4 μm (B) and 2 μm (C). (D) HeLa cells were transfected with Rab5 Q79L-GFP, exposed to 500 μM MnCl₂ for the times indicated, and subsequently stained to detect GPP130. Expression of Rab5 Q79L did not alter the basal localization of GPP130 (data not shown). Bar, 10 μm; insets, 5×. (E) HeLa cells were treated with 500 μM MnCl₂ for 4 h, fixed, and costained to detect GPP130 and Lamp2. Arrowheads indicate overlap between GPP130 and Lamp2. Bar, 4 μm.

Figure 3.

GPP130 is degraded in lysosomes after Mn. (A) HeLa cells were pretreated without or with leupeptin (100 μg/ml) and pepstatin (50 μg/ml) for 24 h (Nicoziani et al., 2000), and then the media were adjusted to 0 or 500 μM MnCl₂ for 8 h. Coimmunostaining was used to detect GPP130 and giantin. Bar, 10 μm. (B) The experiment was also analyzed by immunoblot using antibodies against GPP130 and α-tubulin. (C) Quantitation of GPP130 levels normalized to tubulin levels with GPP130 levels in the absence of Mn as 100% (mean ± SE, n = 3; p < 0.05).

Figure 4.

Depolymerizing microtubules with nocodazole does not block GPP130 degradation. (A) HeLa cells were pretreated for 3 h with or without nocodazole (1 μg/ml; Yadav et al., 2008), and then the media were adjusted to 0 or 500 μM MnCl₂ for 8 h. Coimmunostaining was with antibodies against GPP130 and giantin. Bar, 10 μm. (B and C) The experiment was also analyzed by immunoblot using antibodies against GPP130 and α-tubulin and quantified with GPP130 levels normalized to tubulin and in the absence of Mn expressed as 100% (mean ± SE, n = 3; p > 0.05). (D) HeLa cells were treated with nocodazole, and then the EGF degradation assay was performed. Bar, 10 μm. (E) Quantitation of the mean EGF fluorescence per cell with levels at time 0 expressed as 100% (mean ± SE, n > 12 cells/condition/time point; p < 0.05).

Figure 5.

Mn-induced degradation of GPP130 is Rab7 dependent. (A) HeLa cells were transfected with GFP-tagged Rab7 WT or Rab7 T22N, and 24 h after transfection they were treated with 500 μM MnCl₂ for 8 h or left untreated. Cells were then fixed, stained, and imaged to detect GPP130 and GFP. Bar, 10 μm. (B) Quantitation of the mean GPP130 fluorescence per cell with levels in the absence of Mn normalized to 100% (mean ± SE, n > 12 cells/construct/time point; p < 0.05).

Figure 6.

The GPP130 acidic and cytoplasmic domains are dispensable. (A) Schematic of full-length GPP130 and the deletion constructs lacking the lumenal acidic domain with and without the cytoplasmic domains. Position and residue numbers of cytoplasmic (C), transmembrane (TM), stem, and acidic domains are indicated. “M” indicates the first methionine. (B) HeLa cells were transfected with GPP130_1-247-GFP. Twenty-four hours after transfection, they were exposed to 100 μg/ml cycloheximide for 2 h and then adjusted to 0 or 500 μM MnCl₂ for 2 h. Cells were then fixed, stained, and imaged to detect GPP130 and GFP. Bar, 10 μm. (C) HeLa cells were siRNA-transfected to knockdown endogenous GPP130. After 2 d, the cells were retransfected with an RNAi-immune version of GPP130_1-247-GFP, and after 24 h they were treated with cycloheximide and Mn as described above. The cells were then fixed, stained, and imaged to detect endogenous GPP130 (using anti-GPP130 mAb against acidic domain) and GFP. Bar, 10 μm. (D) HeLa cells were transfected with GPP130_Δ2-11-GFP, treated with cycloheximide and Mn as described above, and processed to detect GPP130 and GFP. Bar, 10 μm.

Figure 7.

Deletions in the stem domain of GPP130 abolish its sensitivity to Mn. (A) Schematic of the deletion constructs used in this figure. All deletions were made on GPP130_1-247-GFP. (B) Cells were transfected with the indicated constructs, and after 24 h they were exposed to cycloheximide for 2 h then adjusted to 500 μM Mn for 2 h. After processing, GPP130 and GFP were imaged in the same cells. Thresholding was used to enhance visualization of endosome with identical thresholds applied to all constructs in a given channel. Bar, 10 μm. (C) Quantitation of the number of GFP endosomes per cell with (gray) and without (black) Mn for each GPP130 construct. GPP130_1-247-GFP was also analyzed after rescue (Res.). The Mn-induced increase in GFP endosomes per cell was statistically significant for GPP130_1-247-GFP, GPP130_1-247-GFP rescue, and GPP130_Δ2-11-GFP (mean ± SE, n > 25 cells for each construct with and without Mn; p < 0.05) but not the other stem deleted constructs (p > 0.05).

Figure 8.

Deletions in the stem domain change the intra-Golgi localization of GPP130. (A) HeLa cells were transfected with the indicated constructs, exposed to cycloheximide for 4 h, and processed to detect endogenous GPP130 (using the anti-acidic domain mAb) and GFP. Line plots to assess overlap between endogenous GPP130 (red) and GFP (green) signals accompany individual immunofluorescence panels. Bar, 10 μm. (B) Plot of the Pearson's coefficient for colocalization between GFP and endogenous GPP130 signals for the indicated constructs. Pearson's coefficient for GPP130_Δ2-11-GFP was significantly greater than all the other constructs (mean ± SE, n > 10 cells/construct; p < 0.05).

Figure 9.

Chimeric GP73-GPP130 constructs are targeted to the cis-Golgi. (A) Schematic of the chimeric constructs. (B) HeLa cells were transfected with chimeric GP73-GPP130 constructs and treated with cycloheximide for 4 h and processed to detect endogenous GPP130 (using anti-acidic domain mAb) and GFP. Line plots for the overlap between endogenous GPP130 (red) and GFP (green) channels are shown. Bar, 10 μm. (C) Plot of the Pearson's coefficient for colocalization between GFP and endogenous GPP130 signals for the indicated constructs. The Pearson's coefficient for the GPP130_Δ2-11-GFP construct is also included for comparison. Except G73-G130_36-175-GFP, there was no significant difference between the Pearson's coefficients for GPP130_Δ2-11-GFP and the chimeric constructs (mean ± SE, n > 10 cells/construct; p > 0.05).

Figure 10.

Chimeric GP73-GPP130 constructs respond to Mn. (A) HeLa cells were transfected with GP73-GPP130 chimeric constructs, and after 24 h they were treated with cycloheximide for 2 h and adjusted to 500 μM Mn for 2 h. The cells were processed to detect GPP130 and GFP. Images depicted are uniformly thresholded to maximize visualization of endosomes. Bar, 10 μm. (B) Quantitation of the number of GFP endosomes per cell with (gray) and without (black) Mn for the indicated GP73-GPP130 chimeric constructs. The Mn-induced increase in GFP endosomes per cell was statistically significant for G73-G130_36-175-GFP, G73-G130_88-247-GFP, and G73-G130_{36-87_176-247}-GFP (mean ± SE, n > 25 cells for each construct with and without Mn; p < 0.05) but not the other chimeric constructs (p > 0.05).