Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and the vacuole

Author:

Grziwa Sophie1,Schäfer Jan-Hannes2ORCID,Nicastro Raffaele3ORCID,Arens Annabel1,De Virgilio Claudio3ORCID,Fröhlich Florian45ORCID,Moeller Arne25ORCID,Gao Jieqiong1ORCID,Langemeyer Lars15ORCID,Ungermann Christian15ORCID

Affiliation:

1. Osnabrück University 1 , Department of Biology/Chemistry, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück , Germany

2. Osnabrück University 2 , Department of Biology/Chemistry, Structural Biology, Barbarastrasse 13, 49076 Osnabrück , Germany

3. University of Fribourg 3 Department of Biology , , Chemin du Musée, CH-1700 Fribourg , Switzerland

4. Osnabrück University 4 , Department of Biology/Chemistry, Molecular Membrane Biology, Barbarastrasse 13, 49076 Osnabrück , Germany

5. Osnabrück University, Center of Cellular Nanoanalytic Osnabrück (CellNanOs) 5 , Barbarastrasse 13, 49076 Osnabrück , Germany

Abstract

ABSTRACT The Saccharomyces cerevisiae casein kinase protein Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates of this protein. We found that endogenously tagged Yck3 localized not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localized to both endosomes and vacuoles, and Yck3 controlled this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, whereas its non-phosphorylatable SA variant strongly localized to endosomes, similar to what was observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying the Rab7-like protein Ypt7. Modeling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which were both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.

Funder

Deutsche Forschungsgemeinschaft

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung

Publisher

The Company of Biologists

Subject

Cell Biology

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