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.

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.

In order to identify cellular genes which interfere with HIV-1 replication in monocyte-derived macrophages (MAC), cells were stimulated with interferon (IFN) or lipopolysaccharide (LPS) leading to a pronounced inhibition of HIV-1 infection in these cells, and the resulting gene expression was analyzed. Using the microarray technology we identified a gene named Stimulated Trans-Acting Factor of 50 kDa (Staf50), which is known to repress the activity of the HIV-1 LTR. Analysis of the Staf50 expression by real-time PCR showed an overexpression in IFNalpha (up to 20-fold) and LPS (up to 10-fold)-stimulated MAC as well as in infected cells (up to 3-fold). For stable overexpression, 293 T cells and primary macrophages were transduced with Staf50-IRES-GFP bicistronic pseudotype viruses. After transduction, 293 T CD4/CCR5 and MAC were infected with HIV-1, and virus replication was monitored by p24 ELISA. Overexpression of Staf50 inhibited the HIV-1 infection between 50% and 90% in 293 T CD4/CCR5 as well as in MAC. Our findings suggest that host genetic effects in combination with viral properties determine the susceptibility of an appropriate target cell for HIV-1 infection as well as the replication potential of the virus in the cell resulting in an overall productive infection.

Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin-protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin-protease matched expression may underlie the imbalanced protease activity observed in pathological states.