Limits...
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.

Show MeSH

Related in: MedlinePlus

Density gradient fractionation of AA and DD VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A), AA (B), and DD (C) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose and the fractions analyzed by Western blotting for HA (A–C, top), and SgII (bottom). The gradients shown were then quantified using NIH Image and the amount of VMAT2 and SgII expressed as a fraction of the total immunoreactivity. Wild-type and AA VMAT2 cofractionate with SgII in heavy fractions. DD also cofractionates with SgII, but has a second peak in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199206&req=5

Figure 7: Density gradient fractionation of AA and DD VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A), AA (B), and DD (C) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose and the fractions analyzed by Western blotting for HA (A–C, top), and SgII (bottom). The gradients shown were then quantified using NIH Image and the amount of VMAT2 and SgII expressed as a fraction of the total immunoreactivity. Wild-type and AA VMAT2 cofractionate with SgII in heavy fractions. DD also cofractionates with SgII, but has a second peak in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.

Mentions: Phosphorylation of the acidic cluster in VMAT2 may also influence targeting to LDCVs. In the case of furin, replacement of phosphorylated serines within the cluster by aspartate to mimic phosphorylation promotes retrieval from LDCVs to the TGN. In contrast, replacement of these serines by alanine to prevent phosphorylation promotes localization to LDCVs (Jones et al. 1995; Dittie et al. 1997). Since serines within the acidic cluster of VMAT2 also undergo phosphorylation by casein kinase 2 (Krantz et al. 1997), we investigated the role of phosphorylation in VMAT2 trafficking to LDCVs by replacing the serines with either alanine (AA mutant) or aspartate (DD mutant). After stable expression of the two constructs in PC12 cells, we compared their localization to SgII by immunofluorescence and density gradient fractionation. Like wild-type VMAT2, the AA mutant colocalizes extensively with SgII at the tips of neuritic processes, and appears at low levels in the cell body (Fig. 6, a–c). By gradient fractionation as well, wild-type and AA VMAT2 colocalize with SgII (Fig. 7 B). The DD mutant also colocalizes with SgII at the tips of processes and in heavy gradient fractions (Fig. 6, d–f, and 7 C), but a significant proportion does not colocalize with SgII. Rather, DD appears partially redistributed to a perinuclear compartment by immunofluorescence and to membranes lighter than LDCVs by gradient fractionation (Fig. 6, d–f, and 7 C).


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)

Density gradient fractionation of AA and DD VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A), AA (B), and DD (C) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose and the fractions analyzed by Western blotting for HA (A–C, top), and SgII (bottom). The gradients shown were then quantified using NIH Image and the amount of VMAT2 and SgII expressed as a fraction of the total immunoreactivity. Wild-type and AA VMAT2 cofractionate with SgII in heavy fractions. DD also cofractionates with SgII, but has a second peak in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199206&req=5

Figure 7: Density gradient fractionation of AA and DD VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A), AA (B), and DD (C) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose and the fractions analyzed by Western blotting for HA (A–C, top), and SgII (bottom). The gradients shown were then quantified using NIH Image and the amount of VMAT2 and SgII expressed as a fraction of the total immunoreactivity. Wild-type and AA VMAT2 cofractionate with SgII in heavy fractions. DD also cofractionates with SgII, but has a second peak in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.
Mentions: Phosphorylation of the acidic cluster in VMAT2 may also influence targeting to LDCVs. In the case of furin, replacement of phosphorylated serines within the cluster by aspartate to mimic phosphorylation promotes retrieval from LDCVs to the TGN. In contrast, replacement of these serines by alanine to prevent phosphorylation promotes localization to LDCVs (Jones et al. 1995; Dittie et al. 1997). Since serines within the acidic cluster of VMAT2 also undergo phosphorylation by casein kinase 2 (Krantz et al. 1997), we investigated the role of phosphorylation in VMAT2 trafficking to LDCVs by replacing the serines with either alanine (AA mutant) or aspartate (DD mutant). After stable expression of the two constructs in PC12 cells, we compared their localization to SgII by immunofluorescence and density gradient fractionation. Like wild-type VMAT2, the AA mutant colocalizes extensively with SgII at the tips of neuritic processes, and appears at low levels in the cell body (Fig. 6, a–c). By gradient fractionation as well, wild-type and AA VMAT2 colocalize with SgII (Fig. 7 B). The DD mutant also colocalizes with SgII at the tips of processes and in heavy gradient fractions (Fig. 6, d–f, and 7 C), but a significant proportion does not colocalize with SgII. Rather, DD appears partially redistributed to a perinuclear compartment by immunofluorescence and to membranes lighter than LDCVs by gradient fractionation (Fig. 6, d–f, and 7 C).

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