Significance In nature, an external input is translated by cellular machinery into a downstream effect through signaling pathways, enabling cells to respond to their environment. Bottom-up synthetic biology aims to re-create cellular organization and function through the creation of “artificial cells” (ACs) through molecular self-assembly. Although the construction of new signaling pathways will enable increasingly responsive ACs, this area is undeveloped; here, we show that multicompartment lipid vesicles are an ideal framework to build a pathway not found in nature. External calcium ions activate internal protein communication, leading to control of cell fluorescence. This highlights the potential of ACs for design and construction of synthetic pathways difficult to reconstitute in existing cells, leading to development of environment-responsive molecular machines in biotechnology.