| Ca2+, a versatile second messenger, regulates many cellular processes. Calcium dynamics is tightly controlled by pumps, channels and exchangers, so that cytosolic Ca2+ levels stay at submicromolar ranges in resting states. Recent studies have identified a new class of pumps, the s&barbelow;ecretory p&barbelow;athway C&barbelow;a 2+-A&barbelow;TPases (SPCAs), first represented by Pmr1 in S. cerevisiae. Haploinsufficiency of Pmr1 homologue in human (hSPCA1) is linked to Hailey-Hailey disease (HHD), a debilitating skin blistering disorder. The early chapters of this work provides the first evidence that hSPCA1 is indeed a Ca2+-ATPase. Expressed in yeast and cultured Chinese Hamster Ovary cells, hSPCA1 resides in the Golgi, complements pmr1 null phenotypes, and transports Ca2+ with high affinity into the Golgi. HHD missence mutants were also expressed in yeast and shown to be inactive, consistent with their roles in causing the skin disorder.; Besides Ca2+, the SPCAs transport Mn2+ with high affinity into the secretory pathway for protein glycosylation. This work characterizes the Pmr1 homologue in C. elegans, and proposes that (i) Golgi, or post-Golgi localization is necessary for manganese tolerance conferred by SPCAs in yeast, and (ii) the N-terminus of SPCAs is important in Golgi localization of the pumps. To identify cellular Mn2+ homeostasis pathways, the S. cerevisiae single-gene knock-out library of 4850 strains was screened for mutants hypersensitive to Mn2+. 75 strains were identified and categorized into different functional groups. These results will be part of a growing database including other genome-wide screens aimed at mapping Ca2+ and Mn 2+ homeostasis pathways.; Perturbations of calcium homeostasis by drugs, mutations, or other stimuli lead to cell death. Amiodarone, a novel fungicide, induces apoptosis-like death in yeast by causing massive calcium influx and release from vacuole. Strikingly, low doses of amiodarone exhibit potent synergism with fungistats. To uncover pathways involved in amiodarone toxicity, the yeast knock-out library was screened for drug hypersensitivity. Some of the mutants identified exhibit pronounced Ca2+ handling defects, confirming the link between AMD toxicity and disruption of Ca2+ homeostasis. Thus, AMD serves as a useful tool in the studies of cellular Ca2+ handling, and promises a new frontier in the fight against fungal infections. |
