2022: Best Master Thesis

Marlene Handl's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Ruprecht-Karls-Universität Heidelberg in the year 2022

Title: From ER to tonoplast: Targeting of VHA-a subunits and visualization of ERtonoplast membrane contact sites

While the V-ATPase is one of the most abundant tonoplast protein complexes it remained to be investigated how its VHA-a subunit dependent sorting is facilitated correctly within the endomembrane system. Within this thesis we identified a PLL amino acid sequence motif that seems to be required for the correct targeting of A. thaliana VHA-a3 and M. polymorpha VHA‑a to the tonoplast.

The vacuole is a multifunctional organelle that occupies up to 90 % of the volume in a plant cell. Most vacuolar functions require a proton gradient which is maintained by the concerted action of Vacuolar-type H⁺-ATPases (V-ATPase) and H⁺-Pyrophosphatases (V-PPase). Targeting of V-ATPase complexes is dependent on the VHA-a isoform incorporated into the membrane integral V₀ subcomplex. In A. thaliana, VHA-a2 and VHA-a3 confer tonoplast localization while VHA-a1 targets the proton pump to the trans-Golgi Network/Early Endosome (TGN/EE) (Dettmer et al., 2006). Recently, a seed plant specific a1-targeting domain (a1-TD) was identified in VHA-a1 that is necessary and sufficient for targeting to the TGN/EE (Lupanga et al., 2020). VHA-a3 is thought to be trafficked to the tonoplast via the COPII-independent provacuolar route (Viotti et al., 2013; Lupanga et al., 2020). As the a1-TD is not conserved in VHA-a3, it remains to be determined if this isoform possesses a sorting signal that targets the membrane protein to the tonoplast. In this thesis, we employed multiple sequence alignments, site-directed mutagenesis, and live imaging to identify a PLL motif that is required for tonoplast sorting of VHA-a3 as well as of the ancestral VHA-a isoform of M. polymorpha. MpVHA-a and VHA-a3 harboring mutations in this motif are mislocalized to the TGN/EE. We further propose that loss of this PLL motif in VHA-a1 was the first step towards the development of a dedicated TGN/EE VHA-a isoform in seed plants.

_{Dettmer, J., Hong-Hermesdorf, A., Stierhof, Y.D., and Schumacher, K. (2006). Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18: 715–730.
Lupanga, U., Röhrich, R., Askani, J., Hilmer, S., Kiefer, C., Krebs, M., Kanazawa, T., Ueda, T., and Schumacher, K. (2020). The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant-specific motif. Elife 9: 1–40.
Viotti, C. et al. (2013). The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole in Arabidopsis. Plant Cell 25: 3434–3449.}

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Marlene Handl conducted this work at the Department of Cell Biology at the Centre for Organismal Studies (COS) Heidelberg in the group of Prof. Dr. Karin Schumacher under the supervision of Prof. Dr. Karin Schumacher and Dr. Upendo Lupanga.