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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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Density gradient fractionation of wild-type and 507* VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A) and 507* (B) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose. Fractions were collected and analyzed by Western blot using the monoclonal HA antibody to detect VMAT2 (A and B, top), and a polyclonal antibody to detect the LDCV marker SgII (bottom). The immunoblots were then digitized and quantified using NIH Image. For each fraction, the amount of VMAT2 and SgII is expressed as a percentage of total immunoreactivity in all the fractions. Wild-type VMAT2 cofractionates with SgII in heavy fractions, whereas 507* cofractionates to a lesser extent with SgII and appears instead in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.
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Figure 2: Density gradient fractionation of wild-type and 507* VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A) and 507* (B) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose. Fractions were collected and analyzed by Western blot using the monoclonal HA antibody to detect VMAT2 (A and B, top), and a polyclonal antibody to detect the LDCV marker SgII (bottom). The immunoblots were then digitized and quantified using NIH Image. For each fraction, the amount of VMAT2 and SgII is expressed as a percentage of total immunoreactivity in all the fractions. Wild-type VMAT2 cofractionates with SgII in heavy fractions, whereas 507* cofractionates to a lesser extent with SgII and appears instead in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.

Mentions: To confirm that deletion of the acidic cluster alters the localization of VMAT2 in PC12 cells, we analyzed the 507* mutant by density gradient fractionation. Equilibrium sedimentation through sucrose, which separates LDCVs from lighter membranes such as SLMVs and endosomes, shows that wild-type VMAT2 colocalizes with SgII in heavy, LDCV-containing fractions (Fig. 2 A), as previously observed (Krantz et al. 2000). Only very small amounts of VMAT2 appear in lighter fractions (Fig. 2 A). In contrast, the 507* mutant colocalizes less well with SgII in heavy fractions and is more prominent in lighter fractions (Fig. 2 B). Equilibrium sedimentation thus confirms the importance of a COOH-terminal acidic cluster for the localization of VMAT2 to LDCVs.


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 wild-type and 507* VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A) and 507* (B) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose. Fractions were collected and analyzed by Western blot using the monoclonal HA antibody to detect VMAT2 (A and B, top), and a polyclonal antibody to detect the LDCV marker SgII (bottom). The immunoblots were then digitized and quantified using NIH Image. For each fraction, the amount of VMAT2 and SgII is expressed as a percentage of total immunoreactivity in all the fractions. Wild-type VMAT2 cofractionates with SgII in heavy fractions, whereas 507* cofractionates to a lesser extent with SgII and appears instead 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 2: Density gradient fractionation of wild-type and 507* VMAT2. Post-nuclear supernatants from PC12 cells stably expressing HA-tagged wild-type (A) and 507* (B) VMAT2 were separated by equilibrium gradient centrifugation through 0.6–1.6 M sucrose. Fractions were collected and analyzed by Western blot using the monoclonal HA antibody to detect VMAT2 (A and B, top), and a polyclonal antibody to detect the LDCV marker SgII (bottom). The immunoblots were then digitized and quantified using NIH Image. For each fraction, the amount of VMAT2 and SgII is expressed as a percentage of total immunoreactivity in all the fractions. Wild-type VMAT2 cofractionates with SgII in heavy fractions, whereas 507* cofractionates to a lesser extent with SgII and appears instead in lighter fractions. The analysis of three different stable cell lines for each construct yielded similar results.
Mentions: To confirm that deletion of the acidic cluster alters the localization of VMAT2 in PC12 cells, we analyzed the 507* mutant by density gradient fractionation. Equilibrium sedimentation through sucrose, which separates LDCVs from lighter membranes such as SLMVs and endosomes, shows that wild-type VMAT2 colocalizes with SgII in heavy, LDCV-containing fractions (Fig. 2 A), as previously observed (Krantz et al. 2000). Only very small amounts of VMAT2 appear in lighter fractions (Fig. 2 A). In contrast, the 507* mutant colocalizes less well with SgII in heavy fractions and is more prominent in lighter fractions (Fig. 2 B). Equilibrium sedimentation thus confirms the importance of a COOH-terminal acidic cluster for the localization of VMAT2 to LDCVs.

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