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An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles.

Waites CL, Mehta A, Tan PK, Thomas G, Edwards RH, Krantz DE - J. Cell Biol. (2001)

Bottom Line: We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs.The motif thus acts as a signal for retention on LDCVs.Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Neuroscience, Department of Neurology, University of California, San Francisco School of Medicine, San Francisco, California 94143-0435, USA.

ABSTRACT
The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.

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The 507* deletion localizes to a BFA-resistant compartment that cofractionates with iLDCVs. (A) PC12 cells stably expressing 507* were incubated without or with 5 μg/ml brefeldin A for 15 min, and then double stained with mouse monoclonal HA antibody and rabbit polyclonal TGN-38 antiserum, followed by anti–mouse antibody conjugated to Cy3 and anti–rabbit antibody conjugated to Cy5. 507* and TGN-38 colocalize in the absence but not the presence of BFA. As indicated by the dispersion of TGN-38, BFA causes fragmentation of the TGN but not the compartment containing 507*. (Insets) Perinuclear staining (arrowheads) at high magnification. Bar, 10 μm. (B) To label SgII in the TGN, cells were incubated for 5 min with 0.5 mCi/ml 35S-sulfate, and then chilled on ice. To allow the labeled SgII to enter iLDCVs, cells were labeled in the same way but incubated at 37°C for an additional 15 min in medium with unlabeled sulfate before harvesting. The resulting post-nuclear supernatants were then separated by velocity sedimentation through 0.3–1.2 M sucrose, and fractions collected from the top of the gradient. Labeled SgII was detected by autoradiography and 507* protein by Western analysis using the monoclonal HA antibody. 507* comigrates with iLDCVs in fractions 2–4 rather than with the TGN in fractions 7–9.
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Figure 4: The 507* deletion localizes to a BFA-resistant compartment that cofractionates with iLDCVs. (A) PC12 cells stably expressing 507* were incubated without or with 5 μg/ml brefeldin A for 15 min, and then double stained with mouse monoclonal HA antibody and rabbit polyclonal TGN-38 antiserum, followed by anti–mouse antibody conjugated to Cy3 and anti–rabbit antibody conjugated to Cy5. 507* and TGN-38 colocalize in the absence but not the presence of BFA. As indicated by the dispersion of TGN-38, BFA causes fragmentation of the TGN but not the compartment containing 507*. (Insets) Perinuclear staining (arrowheads) at high magnification. Bar, 10 μm. (B) To label SgII in the TGN, cells were incubated for 5 min with 0.5 mCi/ml 35S-sulfate, and then chilled on ice. To allow the labeled SgII to enter iLDCVs, cells were labeled in the same way but incubated at 37°C for an additional 15 min in medium with unlabeled sulfate before harvesting. The resulting post-nuclear supernatants were then separated by velocity sedimentation through 0.3–1.2 M sucrose, and fractions collected from the top of the gradient. Labeled SgII was detected by autoradiography and 507* protein by Western analysis using the monoclonal HA antibody. 507* comigrates with iLDCVs in fractions 2–4 rather than with the TGN in fractions 7–9.

Mentions: To determine whether 507* localizes to the TGN or to closely apposed membranes, we treated the stable PC12 transformants with brefeldin A, a fungal derivative that causes fragmentation of the TGN. If expressed in the TGN, 507* should redistribute to the cell periphery after exposure to BFA. BFA indeed collapses TGN-38 as expected, but 507* remains largely perinuclear (Fig. 4 A). Since immature LDCVs (iLDCVs) bud from the TGN and may function as a specialized extension of this sorting compartment, 507* might localize instead to iLDCVs. To test this possibility, we used sulfate labeling of SgII together with gradient fractionation (Tooze and Huttner 1990). Cells were labeled first for 5 min with 35S-sulfate, which incorporates heavily into SgII as it transits the TGN, and then either chilled on ice to prevent the exit of labeled SgII from the TGN, or incubated at 37°C in medium with unlabeled sulfate for an additional 15 min to allow passage of the labeled SgII into iLDCVs. Chase for 15 min does not allow sufficient time for LDCV maturation. We then used velocity gradient fractionation to separate iLDCVs from the TGN. Autoradiography showed the shift of SgII from heavy to light fractions over the 15-min chase (Fig. 4 B), as expected. Western analysis showed that 507* colocalizes with the iLDCV-containing rather than the TGN-containing fractions (Fig. 4 B). Thus, 507* may reside on iLDCVs even though it is greatly reduced on the entire population of LDCVs (Fig. 1 and Fig. 2).


An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles.

Waites CL, Mehta A, Tan PK, Thomas G, Edwards RH, Krantz DE - J. Cell Biol. (2001)

The 507* deletion localizes to a BFA-resistant compartment that cofractionates with iLDCVs. (A) PC12 cells stably expressing 507* were incubated without or with 5 μg/ml brefeldin A for 15 min, and then double stained with mouse monoclonal HA antibody and rabbit polyclonal TGN-38 antiserum, followed by anti–mouse antibody conjugated to Cy3 and anti–rabbit antibody conjugated to Cy5. 507* and TGN-38 colocalize in the absence but not the presence of BFA. As indicated by the dispersion of TGN-38, BFA causes fragmentation of the TGN but not the compartment containing 507*. (Insets) Perinuclear staining (arrowheads) at high magnification. Bar, 10 μm. (B) To label SgII in the TGN, cells were incubated for 5 min with 0.5 mCi/ml 35S-sulfate, and then chilled on ice. To allow the labeled SgII to enter iLDCVs, cells were labeled in the same way but incubated at 37°C for an additional 15 min in medium with unlabeled sulfate before harvesting. The resulting post-nuclear supernatants were then separated by velocity sedimentation through 0.3–1.2 M sucrose, and fractions collected from the top of the gradient. Labeled SgII was detected by autoradiography and 507* protein by Western analysis using the monoclonal HA antibody. 507* comigrates with iLDCVs in fractions 2–4 rather than with the TGN in fractions 7–9.
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Related In: Results  -  Collection

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Figure 4: The 507* deletion localizes to a BFA-resistant compartment that cofractionates with iLDCVs. (A) PC12 cells stably expressing 507* were incubated without or with 5 μg/ml brefeldin A for 15 min, and then double stained with mouse monoclonal HA antibody and rabbit polyclonal TGN-38 antiserum, followed by anti–mouse antibody conjugated to Cy3 and anti–rabbit antibody conjugated to Cy5. 507* and TGN-38 colocalize in the absence but not the presence of BFA. As indicated by the dispersion of TGN-38, BFA causes fragmentation of the TGN but not the compartment containing 507*. (Insets) Perinuclear staining (arrowheads) at high magnification. Bar, 10 μm. (B) To label SgII in the TGN, cells were incubated for 5 min with 0.5 mCi/ml 35S-sulfate, and then chilled on ice. To allow the labeled SgII to enter iLDCVs, cells were labeled in the same way but incubated at 37°C for an additional 15 min in medium with unlabeled sulfate before harvesting. The resulting post-nuclear supernatants were then separated by velocity sedimentation through 0.3–1.2 M sucrose, and fractions collected from the top of the gradient. Labeled SgII was detected by autoradiography and 507* protein by Western analysis using the monoclonal HA antibody. 507* comigrates with iLDCVs in fractions 2–4 rather than with the TGN in fractions 7–9.
Mentions: To determine whether 507* localizes to the TGN or to closely apposed membranes, we treated the stable PC12 transformants with brefeldin A, a fungal derivative that causes fragmentation of the TGN. If expressed in the TGN, 507* should redistribute to the cell periphery after exposure to BFA. BFA indeed collapses TGN-38 as expected, but 507* remains largely perinuclear (Fig. 4 A). Since immature LDCVs (iLDCVs) bud from the TGN and may function as a specialized extension of this sorting compartment, 507* might localize instead to iLDCVs. To test this possibility, we used sulfate labeling of SgII together with gradient fractionation (Tooze and Huttner 1990). Cells were labeled first for 5 min with 35S-sulfate, which incorporates heavily into SgII as it transits the TGN, and then either chilled on ice to prevent the exit of labeled SgII from the TGN, or incubated at 37°C in medium with unlabeled sulfate for an additional 15 min to allow passage of the labeled SgII into iLDCVs. Chase for 15 min does not allow sufficient time for LDCV maturation. We then used velocity gradient fractionation to separate iLDCVs from the TGN. Autoradiography showed the shift of SgII from heavy to light fractions over the 15-min chase (Fig. 4 B), as expected. Western analysis showed that 507* colocalizes with the iLDCV-containing rather than the TGN-containing fractions (Fig. 4 B). Thus, 507* may reside on iLDCVs even though it is greatly reduced on the entire population of LDCVs (Fig. 1 and Fig. 2).

Bottom Line: We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs.The motif thus acts as a signal for retention on LDCVs.Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Neuroscience, Department of Neurology, University of California, San Francisco School of Medicine, San Francisco, California 94143-0435, USA.

ABSTRACT
The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.

Show MeSH
Related in: MedlinePlus