Mechanisms and functions of protein S-acylation modifications at the plant vacuolar membrane
Final Report Abstract
Protein targeting of proteins to the vacuolar membrane mediated by protein S acylation, as demonstrated for several Arabidopsis thaliana Calcineurin B-like (CBL) proteins, is not well understood. Our aim in this project was to get novel, fundamental insights into the underlying requirements for the efficient protein lipid modification and specific protein targeting to the tonoplast of this protein group. Moreover, we also wanted to address the question if other proteins use related or other lipid modification based mechanisms to target to the tonoplast as well. Finally, we tried to determine how the enzymes that can modify such proteins could contribute to this specific mechanism. In this project, we have shown that related CBL proteins, like A. thaliana CBL2 and CBL6, which contain similar N-termini, exhibit differences in the lipid modification mechanism. Especially, we tried to change the CBL2 N-terminus into a CBL6 N-terminus, but we did not achieved tonoplast targeting of this modified CBL2 protein. This importantly showed that the correct structural context in the different CBL2 and CBL6 N-termini are important for efficient lipid modification. We therefore tried to determine the structural determinants within CBL2, which could determine the requirements in lipid modification. We identified a predicted amphipatic helix in CBL2, in which the lipid modification site is directly incorporated. Importantly, when mutations are introduced into the CBL2 N-terminus, which affect amphipatic helix function and formation, the protein is less efficiently lipid modified, and hence impaired in membrane binding and function. However, further studies implied that the amphipatic helix is potentially not directly involved in membrane binding, at least after lipid modification, but likely determines the efficiency of lipid modification. In accordance to that, the inefficient membrane binding of a CBL2 variant, which contains a helix breaker mutation, can be compensated by overexpressing the protein S-acyltransferase (PAT) 10 from Arabidopsis thaliana. Moreover, our studies provide evidence that the lack of basic residues further contributes to the specific tonoplast targeting of CBL2. Based on these structural features, which we have obtained from CBL2, we searched for further proteins with similar properties, and which target to the vacuolar membrane by S-acylation. We identified a new tonoplast targeted protein that, apart from the similarities within the N-terminal region, is not related to CBLs. Surprisingly, in presence of the S- acylation inhibitor 2-Bromopalmitate, this protein is targeted to the plasma membrane, pointing to the possibility that proteins are likely modified at the plasma membrane, and then are targeted to the tonoplast. This localization mechanism could be also involved in the targeting of the flax (Linum usitatissimum) M protein. We have shown here that also this protein contains a CBL2 related N- terminus, which is lipid modified and sufficient for tonoplast targeting. However, in absence of lipid modification, the N-terminus from flax M targets to the plasma membrane as well, indicating that this domain could represent a membrane binding domain in absence of lipid modifications. Importantly, the flax homologous PAT10 enzyme is able to lipid modify the M N-terminus, which would suggest that the S-acylation mechanism of M is related to the lipid modification of A. thaliana CBL2 by PAT10. Surprisingly, the flax PAT10 enzyme is mainly targeted to the plasma membrane, which is in contrast to the localization observed for the homologous A. thaliana PAT10 protein. This further points to our assumption that proteins could first target to the plasma membrane, where they get lipid modified, and then target to the vacuolar membrane. Finally, we were able to establish a screening system for creating mutagenized A. thaliana PAT10 and to identify amino acids that are required for enzyme activity. Importantly, we isolated a mutant PAT10, which is potentially affected in the specific lipid modification mechanism towards Arabidopsis CBL2, while the activity towards another substrate seems to be less compromised. Overall, these studies revealed that both the target as well as the respective enzyme partner indeed contribute together to the specificity in this reaction and targeting mechanism. These novel findings opens now the fundament for future research to address the exact role of the lipid modifications in conjunction with the amphipathic helix formation, and to further determine how the enzyme is contributing to this mechanism. Moreover, the knowledge on the structural requirements will further help us to identify novel target proteins, which contain related lipid modification domains and which use similar or different localization pathways.
Publications
- (2016) “Entwicklung und Anwendung von in vivo Methoden zur Untersuchung der Protein-S-Acylierung am Tonoplasten in Arabidopsis thaliana“
Beckmann, L.
- (2017) N-terminal S-acylation facilitates tonoplast targeting of the calcium sensor CBL6. FEBS Lett., 591, 3745–3756
Zhang, C., Beckmann, L., Kudla, J. and Batistič, O.
(See online at https://doi.org/10.1002/1873-3468.12880)
