Calcium as a regulator of organellar interactions

Diverse cellular membranes and organelles are functionally linked by vesicular compartments, such as the Golgi apparatus, The Trans-Golgi Network, and endosomes. Intriguingly, this also involves chloroplast proteins, a number of which are routed from the Golgi apparatus to the chloroplast and require N- glycosylation that takes place in the Golgi. In animals, many vesicular interactions are regulated by local rises of cytosolic free calcium ([Ca²⁺]cyt), which activate Ca²⁺-binding proteins. These [Ca²⁺]cyt rises are often generated by Ca²⁺ release from the vesicles themselves. Within the vesicles, Ca²⁺ also regulates the modification of protein cargo. Hence, mutants defective in vesicular Ca²⁺ transport are aberrant in organellar communication and in the trafficking of proteins and membranes. In addition, vesicular compartments are a major contributor to global [Ca²⁺]cyt homeostasis of the cell and thereby interfere with Ca²⁺-dependent signalling networks that coordinate many cellular functions. All these roles of vesicular Ca²⁺ are poorly understood in plants, and completely obscure for Golgi-to-chloroplast traffic. Based on our recent identification of various organellar cation transporters as well as our tools to determine Ca²⁺ concentrations on the subcellular scale, this project will analyse Ca²⁺ dynamics in vesicular compartments, the transport proteins that regulate them, and the relevance of vesicular Ca²⁺ homeostasis for the function of these compartments and their interaction with plastids. A number of vesicular transport proteins which we have recently uncovered and which we expect to be of elemental importance in these processes will be functionally characterized for their localization, expression pattern, and substrate spectrum. T-DNA or genome-edited mutant plants for those genes will be subject to phenotypical analyses. To determine concentrations of free Ca²⁺ in cellular compartments and their regulation by those transporters, genetically encoded Ca²⁺ indicators fused to organelle-specific proteins will be employed. The relevance of Ca²⁺- mediated vesicular trafficking for chloroplast protein import will be elucidated by analysing the abundance and glycosylation pattern of chloroplast proteins in mutants altered in vesicular Ca²⁺ homeostasis.