Members of the MATE (Multidrug And Toxin Efflux) family of transporters mediate the transport of organic acid anions that chelate toxic metals (e.g., the soluble aluminum ion Al3+), thereby conferring resistance in poor and marginal soils. Due to the role of the calcineurin B-like protein (CBL)/CBL-interacting protein kinase (CIPK) network in abiotic stress responses in planta, I hypothesized that the CBL-CIPK network modulates MATE function as well. To elucidate these interactions, I focused on the Arabidopsis thaliana CBL-CIPK network and the Arabidopsis thaliana MATE 1 (AtMATE1). Through electrophysiological screening using the Xenopus oocyte heterologous expression system, I have identified the CBL5/CIPK2 complex as a modulator of AtMATE1 activity. The specificity of the protein-protein interactions within the complex and with the transporter was validated by measuring changes in AtMATE1 transport activity in response to structural modifications of the CBL5/CIPK2 complex. Additionally, the targeted alteration of the putative phosphorylation status of AtMATE1 modified its functionality when heterologously co-expressed with the CBL5/CIPK2 complex. Bimolecular fluorescence complementation was used in parallel to validate these protein-protein interactions, together with targeted disruptions of the recombinant proteins in Xenopus oocytes and in Nicotiana benthamiana. Taken together, the results suggest a mechanism by which the CBL5/CIPK2 complex alters the trafficking of AtMATE1 into and out of the plasma membrane. I propose that the CBL5/CIPK2 complex is involved in the stress-resistance response, which includes the phosphorylation of the downstream target protein (AtMATE1), thereby regulating the abiotic stress response in a distinct temporal and spatial manner. In order to better understand the structure, function, and regulation of MATE proteins, novel analytical tools were developed and utilized. These tools include the rapid discovery and generation of single-domain antibodies (nanobodies) that can be used for in vivo tissue and cellular localization, and the development of the SIMPLEx expression system, a method for in vivo solubilization of plant membrane-bound proteins. These tools will further the understanding of the structure, function, and regulation of MATE proteins, in addition to becoming a useful resource for the plant transporter protein community.