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Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

Smaczynska-de Rooij, Iwona I.; Marklew, Christopher J.; Allwood, Ellen G.; Palmer, Sarah E.; Booth, Wesley I.; Mishra, Ritu; Goldberg, Martin W.; Ayscough, Kathryn R.

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Authors

Iwona I. Smaczynska-de Rooij

Christopher J. Marklew

Ellen G. Allwood

Sarah E. Palmer

Wesley I. Booth

Ritu Mishra

Kathryn R. Ayscough



Abstract

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

Citation

Smaczynska-de Rooij, I. I., Marklew, C. J., Allwood, E. G., Palmer, S. E., Booth, W. I., Mishra, R., …Ayscough, K. R. (2016). Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1. Molecular and Cellular Biology, 36(5), 742-755. https://doi.org/10.1128/mcb.00833-15

Journal Article Type Article
Acceptance Date Dec 10, 2015
Online Publication Date Dec 28, 2015
Publication Date Mar 1, 2016
Deposit Date Jun 23, 2017
Publicly Available Date Jun 23, 2017
Journal Molecular and Cellular Biology
Print ISSN 0270-7306
Electronic ISSN 1098-5549
Publisher American Society for Microbiology
Peer Reviewed Peer Reviewed
Volume 36
Issue 5
Pages 742-755
DOI https://doi.org/10.1128/mcb.00833-15

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