Steady-state free Ca2+ in the yeast endoplasmic reticulum reaches only 10 μM and is mainly controlled by the secretory pathway pump Pmr1

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca2+ signaling in eukaryotes. In yeast however, information on intra-organellar Ca2+ concentrations is scarce, despite the demonstrated importance of Ca2+ signals for this microorganism. Here, we directly monitored free Ca2+ in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca2+-sensitive photoprotein aequorin, Ca2+ uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER, At steady-state, the free Ca2+ concentration in the ER lumen was limited to similar to 10 mu M, and ER Ca2+ sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca2+ pumps. In pmr1 null mutants, free Ca2+ in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca2+ uptake activity in yeast.