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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 regulates the intracellular distribution of Sec31. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were harvested 72 h posttransfection and immunoblotted with anti-PP6 antibody (top). Actin (bottom) was used as a loading control. Quantitation of PP6 depletion in three separate experiments is shown in Supplemental Figure S4B. Error bars represent SEM. N = 3. Middle, total (T) lysates from either mock (lanes 1–3) or PP6-depleted (lanes 4–6) cells were centrifuged at 150,000 × g to generate supernatant (S) and pellet (P) fractions. Calnexin was used as a fractionation control. Right, quantitation of the supernatant:pellet ratio of Sec31A from three separate experiments. Error bars represent SEM. N = 3. *p < 0.05, Student's t test. (B) COPI fragments in PP6-depleted cells. COS-7 cells were transfected with mock, PP6 siRNA-08, or PP6 siRNA-08 and pCMV-Myc-hPPP6C-m3-08 (Materials and Methods). The cells were immunostained with anti-COPI antibody (left) and the number of fragmented structures quantified (right). Error bars represent SEM. N > 100 cells in three independent experiments. ***p < 0.001, Student's t test. Scale bar, 20 μm.
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Figure 5: PP6 regulates the intracellular distribution of Sec31. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were harvested 72 h posttransfection and immunoblotted with anti-PP6 antibody (top). Actin (bottom) was used as a loading control. Quantitation of PP6 depletion in three separate experiments is shown in Supplemental Figure S4B. Error bars represent SEM. N = 3. Middle, total (T) lysates from either mock (lanes 1–3) or PP6-depleted (lanes 4–6) cells were centrifuged at 150,000 × g to generate supernatant (S) and pellet (P) fractions. Calnexin was used as a fractionation control. Right, quantitation of the supernatant:pellet ratio of Sec31A from three separate experiments. Error bars represent SEM. N = 3. *p < 0.05, Student's t test. (B) COPI fragments in PP6-depleted cells. COS-7 cells were transfected with mock, PP6 siRNA-08, or PP6 siRNA-08 and pCMV-Myc-hPPP6C-m3-08 (Materials and Methods). The cells were immunostained with anti-COPI antibody (left) and the number of fragmented structures quantified (right). Error bars represent SEM. N > 100 cells in three independent experiments. ***p < 0.001, Student's t test. Scale bar, 20 μm.

Mentions: To address whether PP6 is required for the subcellular distribution of COPII subunits, we used small interfering RNA (siRNA) to deplete cells of PP6 and then examined the distribution of Sec31A. Differential fractionation was performed on COS-7 cells (Figure 5A, middle) transfected with mock or siRNA targeted against PP6 mRNA (Figure 5A, left, and Supplemental Figure S4B), and lysates were separated into supernatant and pellet fractions. As shown in Figure 5A (middle; quantitated on the right), Sec31A distributed between the supernatant and membrane fractions in mock-treated cells (lanes 1–3), whereas in depleted cells it was largely present in the supernatant (lanes 4–6), suggesting that PP6 regulates the recycling of Sec31A on membranes. The loss of PP6 caused a more dispersed punctate localization pattern for the COPI coat (Figure 5B; compare Mock with siPP6-08, described in Materials and Methods; Zeng et al., 2010). This defect was specifically due to the loss of PP6, as it was rescued by the expression of a knockdown-resistant construct of PP6 (Figure 5B; compare siPP6-08 with Rescue). Similar results were obtained with a second siRNA duplex, siPP6-07 (Supplemental Figure S4C).


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 regulates the intracellular distribution of Sec31. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were harvested 72 h posttransfection and immunoblotted with anti-PP6 antibody (top). Actin (bottom) was used as a loading control. Quantitation of PP6 depletion in three separate experiments is shown in Supplemental Figure S4B. Error bars represent SEM. N = 3. Middle, total (T) lysates from either mock (lanes 1–3) or PP6-depleted (lanes 4–6) cells were centrifuged at 150,000 × g to generate supernatant (S) and pellet (P) fractions. Calnexin was used as a fractionation control. Right, quantitation of the supernatant:pellet ratio of Sec31A from three separate experiments. Error bars represent SEM. N = 3. *p < 0.05, Student's t test. (B) COPI fragments in PP6-depleted cells. COS-7 cells were transfected with mock, PP6 siRNA-08, or PP6 siRNA-08 and pCMV-Myc-hPPP6C-m3-08 (Materials and Methods). The cells were immunostained with anti-COPI antibody (left) and the number of fragmented structures quantified (right). Error bars represent SEM. N > 100 cells in three independent experiments. ***p < 0.001, Student's t test. Scale bar, 20 μm.
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Figure 5: PP6 regulates the intracellular distribution of Sec31. (A) Left, COS-7 cells transfected with mock or PP6 siRNA (siPP6-08) were harvested 72 h posttransfection and immunoblotted with anti-PP6 antibody (top). Actin (bottom) was used as a loading control. Quantitation of PP6 depletion in three separate experiments is shown in Supplemental Figure S4B. Error bars represent SEM. N = 3. Middle, total (T) lysates from either mock (lanes 1–3) or PP6-depleted (lanes 4–6) cells were centrifuged at 150,000 × g to generate supernatant (S) and pellet (P) fractions. Calnexin was used as a fractionation control. Right, quantitation of the supernatant:pellet ratio of Sec31A from three separate experiments. Error bars represent SEM. N = 3. *p < 0.05, Student's t test. (B) COPI fragments in PP6-depleted cells. COS-7 cells were transfected with mock, PP6 siRNA-08, or PP6 siRNA-08 and pCMV-Myc-hPPP6C-m3-08 (Materials and Methods). The cells were immunostained with anti-COPI antibody (left) and the number of fragmented structures quantified (right). Error bars represent SEM. N > 100 cells in three independent experiments. ***p < 0.001, Student's t test. Scale bar, 20 μm.
Mentions: To address whether PP6 is required for the subcellular distribution of COPII subunits, we used small interfering RNA (siRNA) to deplete cells of PP6 and then examined the distribution of Sec31A. Differential fractionation was performed on COS-7 cells (Figure 5A, middle) transfected with mock or siRNA targeted against PP6 mRNA (Figure 5A, left, and Supplemental Figure S4B), and lysates were separated into supernatant and pellet fractions. As shown in Figure 5A (middle; quantitated on the right), Sec31A distributed between the supernatant and membrane fractions in mock-treated cells (lanes 1–3), whereas in depleted cells it was largely present in the supernatant (lanes 4–6), suggesting that PP6 regulates the recycling of Sec31A on membranes. The loss of PP6 caused a more dispersed punctate localization pattern for the COPI coat (Figure 5B; compare Mock with siPP6-08, described in Materials and Methods; Zeng et al., 2010). This defect was specifically due to the loss of PP6, as it was rescued by the expression of a knockdown-resistant construct of PP6 (Figure 5B; compare siPP6-08 with Rescue). Similar results were obtained with a second siRNA duplex, siPP6-07 (Supplemental Figure S4C).

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