Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca2+ signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca2+ signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca2+ transporters, combined with manipulations to estimate intracellular Ca2+ storage, we evaluated the contribution of individual transport systems to intracellular Ca2+ homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca2+ homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca2+ uptake and Ca2+ influx across the plasma membrane. In the pmr1 Delta strain, these effects were insensitive to calcineurin activity, independent of Cch1/Midl Ca2+ channels and Pmcl but required Vcxl. By contrast, in the cod1 Delta strain increased vacuolar Ca2+ uptake was not affected by Vcxl deletion but was largely dependent on Pmcl activity. Our analysis further corroborates the distinct roles of Vcxl and Pmc1 in vacuolar Ca2+ uptake and point to the existence of not-yet identified Ca2+ influx pathways. (C) 2015 Elsevier Ltd. All rights reserved.