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Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical

Nature Neuroscience volume 9, pages 1019–1027 (2006)Cite this article

Abstract

Upon exocytosis, synaptic vesicle proteins are released into the plasma membrane and have to be retrieved by compensatory endocytosis. When green fluorescent protein–labeled versions of the vesicle proteins synaptobrevin-2 and synaptotagmin-1 are overexpressed in rat hippocampal neurons, up to 30% are found on axonal membranes under resting conditions. To test whether and to what extent these plasma membrane–stranded proteins participate in exo-endocytic cycling, a new proteolytic approach was used to visualize the fate of newly exocytosed proteins separately from that of the plasma membrane–stranded ones. We found that both pools were mixed and that endocytosed vesicles were largely composed of previously stranded molecules. The degree of nonidentity of vesicular proteins exo- and endocytosed depended on stimulus duration. By using an antibody to the external domain of synaptotagmin-1, we estimated that under physiological conditions a few percent of vesicular proteins were located near the active zone, from where they were preferentially recycled upon stimulation.

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Figure 1: Synaptobrevin-TEV-pHluorin (spH-TEV) is targeted to functional boutons and the plasma membrane–stranded fraction can be effectively cleaved.
Figure 2: Vesicular proteins are lost after fusion.
Figure 3: Vesicular proteins exo- and endocytosed during stimulation are not identical.
Figure 4: Nonidentity is also observed after photobleaching.
Figure 5: Nonidentity is also observed for synaptotagmin-1-TEV-pHluorin.
Figure 6: Stranded pool size is under modulatory control and is about 10% of total pool size under native conditions.
Figure 7: Stranded synaptic vesicle proteins are preferentially recycled.

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Acknowledgements

We are grateful to O. Kochubey for his assistance with data analysis, R. Nehring for advice in molecular biology, E. Neher for support, A.K. Boegle and T. Groemer for critically reading the manuscript, all lab members for fruitful discussions and M. Pilot for expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 523, J.K.), the Human Frontier Science Project (J.K.) and the Boehringer Ingelheim Fonds (M.W.).

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  1. Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen, D-37077, Germany

    Martin Wienisch & Jurgen Klingauf

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  1. Martin Wienisch
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Correspondence to Martin Wienisch.

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Supplementary information

Supplementary Fig. 1

Absolute fluorescence responses from individual boutons. (PDF 73 kb)

Supplementary Fig. 2

Repetitive stimulation post digest and bleaching reveals vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are non-identical. (PDF 204 kb)

Supplementary Fig. 3

For 40 APs also spH-TEV transients of boutons co-overexpressing synaptophysin-mRFP show virtually no recovery postdigest. (PDF 105 kb)

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Wienisch, M., Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat Neurosci 9, 1019–1027 (2006). https://doi.org/10.1038/nn1739

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