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

VMAT2 mutants 507* and DD localize preferentially to immature rather than mature LDCVs. (A) Cells were labeled with 0.2 mCi/ml 35S-sulfate for 6 h, incubated for an additional 12 h with unlabeled sulfate, and PNS separated by velocity sedimentation through 0.3–1.2 M sucrose. Fractions containing mLDCVs were identified by scintillation counting, pooled, and further separated by equilibrium sedimentation through 0.9–1.7 M sucrose. The resulting fractions were collected from the top of the gradient and either submitted to autoradiography to detect radiolabeled SgII or subjected to Western analysis for HA to detect VMAT2. The starting material (PNS) is shown to the right of the fractions. Wild-type VMAT2 cofractionates with labeled SgII in mLDCVs (fractions 9 and 10) and shows enrichment in these fractions relative to the PNS. 507* cofractionates with mLDCVs in fraction 9, but is not enriched relative to the PNS. (B) Cells were labeled with 0.5 mCi/ml 35S-sulfate for 5 min and incubated for an additional 15 min with unlabeled sulfate. After velocity sedimentation, the fractions containing iLDCVs were pooled and further separated by equilibrium sedimentation as described in A, followed by autoradiography for 35S-sulfate, Western analysis for HA, γ-adaptin, transferrin receptor (TfR), and synaptophysin. Both wild-type and 507* VMAT2 cofractionate with labeled SgII in iLDCVs (fractions 7 and 8). Relative to the PNS, 507* is more enriched in iLDCVs than mLDCVs (A). Light membranes such as endosomes and SLMVs do not contaminate the iLDCV fractions containing 35S-SgII. (C) For wt, 507*, AA, and DD VMAT2, three stable cell lines were subjected to the two-step fractionation procedure described above. Peak iLDCV and mLDCV fractions were identified by autoradiography, and the amount of VMAT2 protein in these fractions was measured using NIH Image. The amount of protein on i- and mLDCVs is expressed as a fraction of the total VMAT2 immunoreactivity in all peak fractions. Only the two peak LDCV fractions containing 35S-SgII were used to determine the amount of VMAT2 expressed on i- or mLDCVs. The bars indicate the mean ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199206&req=5

Figure 5: VMAT2 mutants 507* and DD localize preferentially to immature rather than mature LDCVs. (A) Cells were labeled with 0.2 mCi/ml 35S-sulfate for 6 h, incubated for an additional 12 h with unlabeled sulfate, and PNS separated by velocity sedimentation through 0.3–1.2 M sucrose. Fractions containing mLDCVs were identified by scintillation counting, pooled, and further separated by equilibrium sedimentation through 0.9–1.7 M sucrose. The resulting fractions were collected from the top of the gradient and either submitted to autoradiography to detect radiolabeled SgII or subjected to Western analysis for HA to detect VMAT2. The starting material (PNS) is shown to the right of the fractions. Wild-type VMAT2 cofractionates with labeled SgII in mLDCVs (fractions 9 and 10) and shows enrichment in these fractions relative to the PNS. 507* cofractionates with mLDCVs in fraction 9, but is not enriched relative to the PNS. (B) Cells were labeled with 0.5 mCi/ml 35S-sulfate for 5 min and incubated for an additional 15 min with unlabeled sulfate. After velocity sedimentation, the fractions containing iLDCVs were pooled and further separated by equilibrium sedimentation as described in A, followed by autoradiography for 35S-sulfate, Western analysis for HA, γ-adaptin, transferrin receptor (TfR), and synaptophysin. Both wild-type and 507* VMAT2 cofractionate with labeled SgII in iLDCVs (fractions 7 and 8). Relative to the PNS, 507* is more enriched in iLDCVs than mLDCVs (A). Light membranes such as endosomes and SLMVs do not contaminate the iLDCV fractions containing 35S-SgII. (C) For wt, 507*, AA, and DD VMAT2, three stable cell lines were subjected to the two-step fractionation procedure described above. Peak iLDCV and mLDCV fractions were identified by autoradiography, and the amount of VMAT2 protein in these fractions was measured using NIH Image. The amount of protein on i- and mLDCVs is expressed as a fraction of the total VMAT2 immunoreactivity in all peak fractions. Only the two peak LDCV fractions containing 35S-SgII were used to determine the amount of VMAT2 expressed on i- or mLDCVs. The bars indicate the mean ± SEM (n = 3).

Mentions: The presence of 507* on iLDCVs despite a low proportion on LDCVs raises the possibility that the mutant is removed during LDCV maturation. To determine whether 507* indeed resides on iLDCVs, we again used the labeling of SgII with 35S-sulfate followed by gradient fractionation (Dittie et al. 1997; Blagoveshchenskaya et al. 1999). Labeling for 5 min followed by incubation for an additional 15 min with unlabeled sulfate allows the labeled SgII to enter iLDCVs, but not mature LDCVs. Labeling for 6 h followed by 12 h of chase allows sufficient time for the maturation of LDCVs containing labeled SgII. To separate iLDCVs from mLDCVs, and hence determine the fate of VMAT2 during LDCV maturation, we used sequential velocity and equilibrium sedimentation through sucrose. Autoradiography for 35S-labeled SgII identified the fractions containing either iLDCVs or mLDCVs, and Western blotting identified those that contain VMAT2. Wild-type VMAT2 colocalizes with labeled SgII in both i- and mLDCV fractions (Fig. 5A and Fig. B). In contrast, 507* is barely detectable in mLDCV-containing fractions, but colocalizes with labeled SgII in iLDCV-containing gradient fractions (Fig. 5A and Fig. B). These iLDCV fractions do not contain detectable amounts of other membranes, including endosomes and SLMVs (Fig. 5 B), enabling us to distinguish between the VMAT2 on iLDCVs and on other membranes. To quantify these observations, we studied three stable cell lines each for wild-type and 507* VMAT2, and compared the amounts of protein on i- and mLDCVs. Using autoradiography to identify the peak iLDCV and mLDCV fractions, we determined the amount of VMAT2 in these fractions by densitometry and expressed the amount in either iLDCVs or mLDCVs as a fraction of total VMAT2 in all peak fractions. The analysis shows that more than half of wild-type VMAT2 in LDCVs localizes to mLDCVs, whereas only a small fraction (∼10%) of 507* resides on these membranes (Fig. 5 C). In contrast, a substantially larger proportion of 507* localizes to iLDCVs relative to wild-type VMAT2. The quantitation of iLDCVs specifically excludes VMAT2 that does not cofractionate with the peak of SgII, focusing on the protein in iLDCVs rather than that in endosomes or SLMVs. A substantial proportion of the 507* that enters the regulated secretory pathway thus appears to be removed from LDCVs during maturation, indicating a role for the acidic cluster in retention on mLDCVs.


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)

VMAT2 mutants 507* and DD localize preferentially to immature rather than mature LDCVs. (A) Cells were labeled with 0.2 mCi/ml 35S-sulfate for 6 h, incubated for an additional 12 h with unlabeled sulfate, and PNS separated by velocity sedimentation through 0.3–1.2 M sucrose. Fractions containing mLDCVs were identified by scintillation counting, pooled, and further separated by equilibrium sedimentation through 0.9–1.7 M sucrose. The resulting fractions were collected from the top of the gradient and either submitted to autoradiography to detect radiolabeled SgII or subjected to Western analysis for HA to detect VMAT2. The starting material (PNS) is shown to the right of the fractions. Wild-type VMAT2 cofractionates with labeled SgII in mLDCVs (fractions 9 and 10) and shows enrichment in these fractions relative to the PNS. 507* cofractionates with mLDCVs in fraction 9, but is not enriched relative to the PNS. (B) Cells were labeled with 0.5 mCi/ml 35S-sulfate for 5 min and incubated for an additional 15 min with unlabeled sulfate. After velocity sedimentation, the fractions containing iLDCVs were pooled and further separated by equilibrium sedimentation as described in A, followed by autoradiography for 35S-sulfate, Western analysis for HA, γ-adaptin, transferrin receptor (TfR), and synaptophysin. Both wild-type and 507* VMAT2 cofractionate with labeled SgII in iLDCVs (fractions 7 and 8). Relative to the PNS, 507* is more enriched in iLDCVs than mLDCVs (A). Light membranes such as endosomes and SLMVs do not contaminate the iLDCV fractions containing 35S-SgII. (C) For wt, 507*, AA, and DD VMAT2, three stable cell lines were subjected to the two-step fractionation procedure described above. Peak iLDCV and mLDCV fractions were identified by autoradiography, and the amount of VMAT2 protein in these fractions was measured using NIH Image. The amount of protein on i- and mLDCVs is expressed as a fraction of the total VMAT2 immunoreactivity in all peak fractions. Only the two peak LDCV fractions containing 35S-SgII were used to determine the amount of VMAT2 expressed on i- or mLDCVs. The bars indicate the mean ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: VMAT2 mutants 507* and DD localize preferentially to immature rather than mature LDCVs. (A) Cells were labeled with 0.2 mCi/ml 35S-sulfate for 6 h, incubated for an additional 12 h with unlabeled sulfate, and PNS separated by velocity sedimentation through 0.3–1.2 M sucrose. Fractions containing mLDCVs were identified by scintillation counting, pooled, and further separated by equilibrium sedimentation through 0.9–1.7 M sucrose. The resulting fractions were collected from the top of the gradient and either submitted to autoradiography to detect radiolabeled SgII or subjected to Western analysis for HA to detect VMAT2. The starting material (PNS) is shown to the right of the fractions. Wild-type VMAT2 cofractionates with labeled SgII in mLDCVs (fractions 9 and 10) and shows enrichment in these fractions relative to the PNS. 507* cofractionates with mLDCVs in fraction 9, but is not enriched relative to the PNS. (B) Cells were labeled with 0.5 mCi/ml 35S-sulfate for 5 min and incubated for an additional 15 min with unlabeled sulfate. After velocity sedimentation, the fractions containing iLDCVs were pooled and further separated by equilibrium sedimentation as described in A, followed by autoradiography for 35S-sulfate, Western analysis for HA, γ-adaptin, transferrin receptor (TfR), and synaptophysin. Both wild-type and 507* VMAT2 cofractionate with labeled SgII in iLDCVs (fractions 7 and 8). Relative to the PNS, 507* is more enriched in iLDCVs than mLDCVs (A). Light membranes such as endosomes and SLMVs do not contaminate the iLDCV fractions containing 35S-SgII. (C) For wt, 507*, AA, and DD VMAT2, three stable cell lines were subjected to the two-step fractionation procedure described above. Peak iLDCV and mLDCV fractions were identified by autoradiography, and the amount of VMAT2 protein in these fractions was measured using NIH Image. The amount of protein on i- and mLDCVs is expressed as a fraction of the total VMAT2 immunoreactivity in all peak fractions. Only the two peak LDCV fractions containing 35S-SgII were used to determine the amount of VMAT2 expressed on i- or mLDCVs. The bars indicate the mean ± SEM (n = 3).
Mentions: The presence of 507* on iLDCVs despite a low proportion on LDCVs raises the possibility that the mutant is removed during LDCV maturation. To determine whether 507* indeed resides on iLDCVs, we again used the labeling of SgII with 35S-sulfate followed by gradient fractionation (Dittie et al. 1997; Blagoveshchenskaya et al. 1999). Labeling for 5 min followed by incubation for an additional 15 min with unlabeled sulfate allows the labeled SgII to enter iLDCVs, but not mature LDCVs. Labeling for 6 h followed by 12 h of chase allows sufficient time for the maturation of LDCVs containing labeled SgII. To separate iLDCVs from mLDCVs, and hence determine the fate of VMAT2 during LDCV maturation, we used sequential velocity and equilibrium sedimentation through sucrose. Autoradiography for 35S-labeled SgII identified the fractions containing either iLDCVs or mLDCVs, and Western blotting identified those that contain VMAT2. Wild-type VMAT2 colocalizes with labeled SgII in both i- and mLDCV fractions (Fig. 5A and Fig. B). In contrast, 507* is barely detectable in mLDCV-containing fractions, but colocalizes with labeled SgII in iLDCV-containing gradient fractions (Fig. 5A and Fig. B). These iLDCV fractions do not contain detectable amounts of other membranes, including endosomes and SLMVs (Fig. 5 B), enabling us to distinguish between the VMAT2 on iLDCVs and on other membranes. To quantify these observations, we studied three stable cell lines each for wild-type and 507* VMAT2, and compared the amounts of protein on i- and mLDCVs. Using autoradiography to identify the peak iLDCV and mLDCV fractions, we determined the amount of VMAT2 in these fractions by densitometry and expressed the amount in either iLDCVs or mLDCVs as a fraction of total VMAT2 in all peak fractions. The analysis shows that more than half of wild-type VMAT2 in LDCVs localizes to mLDCVs, whereas only a small fraction (∼10%) of 507* resides on these membranes (Fig. 5 C). In contrast, a substantially larger proportion of 507* localizes to iLDCVs relative to wild-type VMAT2. The quantitation of iLDCVs specifically excludes VMAT2 that does not cofractionate with the peak of SgII, focusing on the protein in iLDCVs rather than that in endosomes or SLMVs. A substantial proportion of the 507* that enters the regulated secretory pathway thus appears to be removed from LDCVs during maturation, indicating a role for the acidic cluster in retention on mLDCVs.

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