No abstract

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects ofpmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions ‐469, ‐245 and ‐185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.

We report a structural and functional analysis of the PMR2 gene cluster in yeast. We found that several strains of Saccharomyces cerevisiae contain multiple PMR2 genes repeated in tandem, whereas most phylogenetically related yeasts appear to possess only a single PMR2 gene. This unusual tandem array of nearly identical genes encodes putative ion pumps involved in Na+ tolerance. Pmr2a and Pmr2b, the proteins encoded by the first two repeats, differ by only 13 amino acid exchanges. Both proteins share localization to the plasma membrane, but represent distinct isoforms of a putative Na+ pump. When expressed under identical conditions in vivo, Pmr2a and Pmr2b cause different tolerances to Na+ and Li+. Finally, we show that the Na+ tolerance mediated through these pumps is regulated by calmodulin via a calcineurin‐independent mechanism which activates the Pmr2 ion pumps post‐transcriptionally.

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca 2⍣ signaling in eukaryotes. In yeast however, information on intra-organellar Ca 2⍣ concentrations is scarce, despite the demonstrated importance of Ca 2⍣ signals for this microorganism. Here, we directly monitored free Ca 2⍣ in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca 2⍣ -sensitive photoprotein aequorin. Ca 2⍣ uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steadystate, the free Ca 2⍣ concentration in the ER lumen was limited to~10 μM, and ER Ca 2⍣ sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca 2⍣ pumps. In pmr1 null mutants, free Ca 2⍣ 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 Ca 2⍣ uptake activity in yeast.