Functional Expression in Yeast of the Human Secretory Pathway Ca2+, Mn2+-ATPase Defective in Hailey-Hailey Disease

Abstract

The discovery and biochemical characterization of the secretory pathway Ca 2؉ -ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens.

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

“…In conjunction with the Mn 2ϩ -tolerant phenotypes described above, these data suggest that high-affinity Mn 2ϩ transport is a unique property of the secretory pathway pumps. Green fluorescent protein (GFP)-tagged hSPCA1 localized to the Golgi in yeast, demonstrating the elegant simplicity yet effectiveness of functional complementation (83). Taken together, these data led us to conclude that hSPCA1 is a functional ortholog of yeast Pmr1.…”

“…Expression of a high-affinity Mn 2ϩ pump effectively compartmentalizes the toxic ion and rescues cell growth. Interestingly, hSPCA1, but not rabbit SERCA1 or human PMCA4, was seen to complement the Mn 2ϩ -sensitive growth defect of the pmr1 strain (83).…”

“…Expression of a high-affinity Ca 2ϩ pump rescues the cells from Ca 2ϩ starvation. It was observed that hSPCA1 fully complemented the BAPTA hypersensitivity of K616, as did heterologous expression of mammalian PMCA and SERCA pumps (83). Mn 2ϩ hypersensitivity of the pmr1 mutant results from the toxicity of excessive cellular Mn 2ϩ ; for example, Mn 2ϩ is known to increase the error rate of DNA polymerases and to interfere with Mg 2ϩ -binding enzymes (9).…”

“…As a microbial expression system, Saccharomyces offers more advantages than Escherichia coli, since exogenous proteins may be targeted to membrane-bound compartments that are readily isolated for biochemical experiments (23,78,83). It also surpasses cultured insect and mammalian cell lines with simple and inexpensive growth conditions and a shorter cell cycle.…”

“…Pmr1 homologs are now known to be ubiquitous in all higher eukaryotes, with the notable exception of plants, and much of what we know about the structure and physiological role of these transporters comes from studies in yeast. More recently, these studies have extended to SPCA homologs from humans (5,27,83), Caenorhabditis elegans (13,87), and rats (Ref. 69; reviewed in Ref.…”