The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Consumers as mental health providers: First-person accounts of benefits and limitations

Community support programs are increasingly establishing paid service positions designated exclusively for consumers. Project WINS (Work Incentives and Needs Study), a hybrid case management-vocational program for individuals with severe mental illness, used consumers as peer support specialists (PSSs) to supplement professional roles. Semistructured interviews were conducted with PSSs about 12 months after their employment ended. They identified substantial personal benefits specific to consumer-designated roles (e.g., a “safe” employment setting with accommodations) and general benefits from employment. Problems described were just as numerous, encompassing attitudes toward assigned peers and costs to their own well-being. Critical commentary addressed program operations (structure, supervision, and training needs) and problems in the mental health system. The authors discuss the changed sense of self that service provider roles can create for consumers and suggest that mental health administrators provide anticipatory socialization for this service innovation throughout their agencies and ongoing supports for consumers in their new roles.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45767/1/11414_2005_Article_BF02287510.pd

A Genome-Wide Immunodetection Screen in S. cerevisiae Uncovers Novel Genes Involved in Lysosomal Vacuole Function and Morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events

Multiple Means to the Same End: The Genetic Basis of Acquired Stress Resistance in Yeast

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H2O2). Surprisingly, there was little overlap in the genes required for acquisition of H2O2 tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H2O2 in each case. Integrative network analysis of these results with respect to protein–protein interactions, synthetic–genetic interactions, and functional annotations identified many processes not previously linked to H2O2 tolerance. We tested and present several models that explain the lack of overlap in genes required for H2O2 tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance

Quantifying the relationship between sequence and three-dimensional structure conservation in RNA

<p>Abstract</p> <p>Background</p> <p>In recent years, the number of available RNA structures has rapidly grown reflecting the increased interest on RNA biology. Similarly to the studies carried out two decades ago for proteins, which gave the fundamental grounds for developing comparative protein structure prediction methods, we are now able to quantify the relationship between sequence and structure conservation in RNA.</p> <p>Results</p> <p>Here we introduce an all-against-all sequence- and three-dimensional (3D) structure-based comparison of a representative set of RNA structures, which have allowed us to quantitatively confirm that: (i) there is a measurable relationship between sequence and structure conservation that weakens for alignments resulting in below 60% sequence identity, (ii) evolution tends to conserve more RNA structure than sequence, and (iii) there is a twilight zone for RNA homology detection.</p> <p>Discussion</p> <p>The computational analysis here presented quantitatively describes the relationship between sequence and structure for RNA molecules and defines a twilight zone region for detecting RNA homology. Our work could represent the theoretical basis and limitations for future developments in comparative RNA 3D structure prediction.</p

Three-dimensional RNA structure refinement by hydroxyl radical probing

Molecular modeling guided by experimentally-derived structural information is an attractive approach for three-dimensional structure determination of complex RNAs that are not amenable to study by high-resolution methods. Hydroxyl radical probing (HRP), performed routinely in many laboratories, provides a measure of solvent accessibility at individual nucleotides. HRP measurements have, to date, only been used to evaluate RNA models qualitatively. Here, we report development of a quantitative structure refinement approach using HRP measurements to drive discrete molecular dynamics simulations for RNAs ranging in size from 80 to 230 nucleotides. HRP reactivities were first used to identify RNAs that form extensive helical packing interactions. For these RNAs, we achieved highly significant structure predictions, given inputs of RNA sequence and base pairing. This HRP-directed tertiary structure refinement approach generates robust structural hypotheses useful for guiding explorations of structure-function interrelationships in RNA