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Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits.

Bhandari D, Zhang J, Menon S, Lord C, Chen S, Helm JR, Thorsen K, Corbett KD, Hay JC, Ferro-Novick S - Mol. Biol. Cell (2013)

Bottom Line: Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo.In vitro, Sit4p dephosphorylates COPII coat subunits.Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

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PP6 is required for ER-to-Golgi traffic in mammalian cells. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were transfected with tsO45VSV-G-GFP and incubated at 40°C for 20 h. Cells were then treated with cycloheximide (100 μg/ml final concentration) for 30 min and shifted to 32°C for 0, 5, and 10 min, permeabilized with 0.1% Triton X-100, and immunostained with anti-GM130 antibody (red). Right, data shown on the left quantitated as described in Thayanidhi et al. (2010). Error bars represent the SEM. N = 3. More than 20 cells were examined for each time point in three separate experiments in which ∼89% of the PP6 was depleted. ***p < 0.001, Student's t test. Scale bar, 20 μm. (B) COS-7 cells were transfected with PP6 siRNA or mock transfected with Lipofectamine reagent alone and grown for 3 d. Cells were then either fixed directly (untreated) or incubated 1 h with BFA, followed by fixation (0-min washout) or recovery in medium without BFA for 75 min before fixation (75-min washout). Quantitation of pronounced giantin Golgi intensity (Materials and Methods) from >125 randomly selected cells for each plotted value. Y-axis value = (Golgi intensity at 75 min washout − Golgi intensity at 0-min washout)/(Golgi intensity of mock cells − Golgi intensity at 0 min) × 100%. Error bars represent SEM. Results are from a single representative experiment. The experiment was performed three times with similar outcomes. **p < 0.01, Student's t test. Knockdown of PP6 was 90% as assessed by immunoblotting of duplicate coverslips.
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Figure 7: PP6 is required for ER-to-Golgi traffic in mammalian cells. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were transfected with tsO45VSV-G-GFP and incubated at 40°C for 20 h. Cells were then treated with cycloheximide (100 μg/ml final concentration) for 30 min and shifted to 32°C for 0, 5, and 10 min, permeabilized with 0.1% Triton X-100, and immunostained with anti-GM130 antibody (red). Right, data shown on the left quantitated as described in Thayanidhi et al. (2010). Error bars represent the SEM. N = 3. More than 20 cells were examined for each time point in three separate experiments in which ∼89% of the PP6 was depleted. ***p < 0.001, Student's t test. Scale bar, 20 μm. (B) COS-7 cells were transfected with PP6 siRNA or mock transfected with Lipofectamine reagent alone and grown for 3 d. Cells were then either fixed directly (untreated) or incubated 1 h with BFA, followed by fixation (0-min washout) or recovery in medium without BFA for 75 min before fixation (75-min washout). Quantitation of pronounced giantin Golgi intensity (Materials and Methods) from >125 randomly selected cells for each plotted value. Y-axis value = (Golgi intensity at 75 min washout − Golgi intensity at 0-min washout)/(Golgi intensity of mock cells − Golgi intensity at 0 min) × 100%. Error bars represent SEM. Results are from a single representative experiment. The experiment was performed three times with similar outcomes. **p < 0.01, Student's t test. Knockdown of PP6 was 90% as assessed by immunoblotting of duplicate coverslips.

Mentions: To address whether VSV-G traffic was delayed between the ER and Golgi complex in depleted cells, we examined the intracellular pool of VSV-G at 0, 5, and 10 min after shifting the cells to 32°C. At 0 min, VSV-G resided in the ER in mock and depleted cells. By 5 min, some VSV-G reached the Golgi in the mock-treated cells, but delivery was delayed in the depleted cells. By 10 min, most of the VSV-G had reached the Golgi in mock-treated but not depleted cells (Figure 7A, left). Quantitation of the increasing ratio of juxtanuclear to peripheral VSV-G (Figure 7A, right) revealed a significant delay in the depleted cells at both 5 and 10 min. Similar results were obtained with a second siRNAi duplex, siPP6-07 (unpublished observations). During these experiments we noted that GM130 did not fragment in the PP6-depleted cells. Other integral membrane Golgi markers discussed later also did not show any fragmentation. Thus depletion of PP6 may indirectly affect COPI dynamics by disrupting ER-to-Golgi traffic without significantly affecting Golgi morphology.


Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits.

Bhandari D, Zhang J, Menon S, Lord C, Chen S, Helm JR, Thorsen K, Corbett KD, Hay JC, Ferro-Novick S - Mol. Biol. Cell (2013)

PP6 is required for ER-to-Golgi traffic in mammalian cells. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were transfected with tsO45VSV-G-GFP and incubated at 40°C for 20 h. Cells were then treated with cycloheximide (100 μg/ml final concentration) for 30 min and shifted to 32°C for 0, 5, and 10 min, permeabilized with 0.1% Triton X-100, and immunostained with anti-GM130 antibody (red). Right, data shown on the left quantitated as described in Thayanidhi et al. (2010). Error bars represent the SEM. N = 3. More than 20 cells were examined for each time point in three separate experiments in which ∼89% of the PP6 was depleted. ***p < 0.001, Student's t test. Scale bar, 20 μm. (B) COS-7 cells were transfected with PP6 siRNA or mock transfected with Lipofectamine reagent alone and grown for 3 d. Cells were then either fixed directly (untreated) or incubated 1 h with BFA, followed by fixation (0-min washout) or recovery in medium without BFA for 75 min before fixation (75-min washout). Quantitation of pronounced giantin Golgi intensity (Materials and Methods) from >125 randomly selected cells for each plotted value. Y-axis value = (Golgi intensity at 75 min washout − Golgi intensity at 0-min washout)/(Golgi intensity of mock cells − Golgi intensity at 0 min) × 100%. Error bars represent SEM. Results are from a single representative experiment. The experiment was performed three times with similar outcomes. **p < 0.01, Student's t test. Knockdown of PP6 was 90% as assessed by immunoblotting of duplicate coverslips.
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Related In: Results  -  Collection

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Figure 7: PP6 is required for ER-to-Golgi traffic in mammalian cells. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were transfected with tsO45VSV-G-GFP and incubated at 40°C for 20 h. Cells were then treated with cycloheximide (100 μg/ml final concentration) for 30 min and shifted to 32°C for 0, 5, and 10 min, permeabilized with 0.1% Triton X-100, and immunostained with anti-GM130 antibody (red). Right, data shown on the left quantitated as described in Thayanidhi et al. (2010). Error bars represent the SEM. N = 3. More than 20 cells were examined for each time point in three separate experiments in which ∼89% of the PP6 was depleted. ***p < 0.001, Student's t test. Scale bar, 20 μm. (B) COS-7 cells were transfected with PP6 siRNA or mock transfected with Lipofectamine reagent alone and grown for 3 d. Cells were then either fixed directly (untreated) or incubated 1 h with BFA, followed by fixation (0-min washout) or recovery in medium without BFA for 75 min before fixation (75-min washout). Quantitation of pronounced giantin Golgi intensity (Materials and Methods) from >125 randomly selected cells for each plotted value. Y-axis value = (Golgi intensity at 75 min washout − Golgi intensity at 0-min washout)/(Golgi intensity of mock cells − Golgi intensity at 0 min) × 100%. Error bars represent SEM. Results are from a single representative experiment. The experiment was performed three times with similar outcomes. **p < 0.01, Student's t test. Knockdown of PP6 was 90% as assessed by immunoblotting of duplicate coverslips.
Mentions: To address whether VSV-G traffic was delayed between the ER and Golgi complex in depleted cells, we examined the intracellular pool of VSV-G at 0, 5, and 10 min after shifting the cells to 32°C. At 0 min, VSV-G resided in the ER in mock and depleted cells. By 5 min, some VSV-G reached the Golgi in the mock-treated cells, but delivery was delayed in the depleted cells. By 10 min, most of the VSV-G had reached the Golgi in mock-treated but not depleted cells (Figure 7A, left). Quantitation of the increasing ratio of juxtanuclear to peripheral VSV-G (Figure 7A, right) revealed a significant delay in the depleted cells at both 5 and 10 min. Similar results were obtained with a second siRNAi duplex, siPP6-07 (unpublished observations). During these experiments we noted that GM130 did not fragment in the PP6-depleted cells. Other integral membrane Golgi markers discussed later also did not show any fragmentation. Thus depletion of PP6 may indirectly affect COPI dynamics by disrupting ER-to-Golgi traffic without significantly affecting Golgi morphology.

Bottom Line: Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo.In vitro, Sit4p dephosphorylates COPII coat subunits.Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

Show MeSH
Related in: MedlinePlus