A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

Breker, Michal; Gymrek, Melissa; Schuldiner, Maya

doi:10.1083/jcb.201301120

Cited by 212 publications

(219 citation statements)

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“…The Ino substrate for Pis1 is synthesized by the Ino-3-phosphate synthase Ino1 and the phosphatases Inm1 and Inm2, all of which are cytosolic [28]. Similarly, CDP-DAG is produced by Cds1, an integral membrane protein of the ER [29], for which TOPCONS predictors unanimously locate most highly conserved polar residues to the cytosolic side (Fig.…”

Section: Discussionmentioning

confidence: 99%

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

Bochud

Conzelmann

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.

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Section: Discussionmentioning

confidence: 99%

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

Bochud

Conzelmann

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…To integrate the GFPOm14 into a library of deletions in all nonessential genes (24) and a library of hypomorphic alleles of all essential genes (23), we used SGA to attain nearly 6,000 haploid strains, with each expressing GFP-OM14 on the background of one mutation. The strains were then visualized using a previously described high-content microscopy setup (27). Normal mitochondrial staining was observed in the vast majority (97.8%) of the inspected mutated strains (see, for example, Fig.…”

Section: Gfp-om14 Serves As a Model Protein For Correct Membrane Insementioning

confidence: 99%

“…High-throughput screens were performed with a system previously described (26,27). The microscopy for follow-up analysis was performed using an Olympus IX71 microscope controlled by Delta Vision SoftWoRx 3.5.1 software with either 60ϫ or 100ϫ oil lenses.…”

Section: Construction Of Om14 Variants and Yeast Strainsmentioning

confidence: 99%

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

Sauerwald

Jores

Eisenberg-Bord

et al. 2015

Molecular and Cellular Biology

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A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1⌬ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins.

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“…omprehensive localization studies have provided a wealth of information about the functions of different Saccharomyces cerevisiae proteins (1,2). To date, most studies have examined protein localization only during mitotic growth in rich medium.…”

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confidence: 99%

A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast

et al. 2014

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During ascospore formation in Saccharomyces cerevisiae, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1, but not ADY3. Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.C omprehensive localization studies have provided a wealth of information about the functions of different Saccharomyces cerevisiae proteins (1, 2). To date, most studies have examined protein localization only during mitotic growth in rich medium. The localization of proteins that are expressed only under specific conditions has not been systematically examined. Moreover, constitutively expressed proteins can also be relocalized under different conditions. Many examples of such changes in distribution occur when yeast cells undergo sporulation (3-6).When diploid yeast cells are starved for nitrogen in the presence of a nonfermentable carbon source, they exit the mitotic cycle and enter the developmental program of meiosis and sporulation (7). Spores are created in an unusual cell division in which membranes are formed de novo in the cytosol and enclose each of the daughter nuclei produced by meiosis. These prospore membranes initially form on the cytoplasmic face of each of the four spindle pole bodies (SPBs) present in meiosis II. The membranes then expand beyond the SPBs to engulf the nuclei. As they do so, their shape is constrained by membrane-associated protein complexes.One of these membrane-associated complexes, the leadingedge protein complex (LEP), composed of the proteins Ssp1, Ady3, and Don1, forms a ring structure at the lip of the prospore membrane (8-10). The LEP is organized in a stratified fashion, with SSP1 being required for the localization of Ady3 and Don1 and ADY3 being required for the localization of Don1. The LEP helps to control the shape of the prospore membrane and is proposed to exert an outward for...

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A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

Abstract: Unprecedented proteome plasticity in response to stress in yeast is revealed using a novel screening platform that allows tracking of protein localization and abundance at single-cell resolution.

Cited by 212 publications

References 64 publications

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast

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