Overview of Subcellular Fractionation Procedures for the Yeast<i>Saccharomyces cerevisiae</i>

Rieder, Stephanie E.; Emr, Scott D.

doi:10.1002/0471143030.cb0307s07

Cited by 15 publications

(13 citation statements)

References 57 publications

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“…To determine whether PalB:(HA)3, expressed at physiological levels, is associated with membranes, we performed subcellular fractionation studies. Protoplast lysates were separated into 13,000 ϫ g pellet (P13) and supernatant fractions, and the supernatant further separated into 100,000 ϫ g soluble (S100) and insoluble (P100) fractions, as described for yeast (35,48,49). In agreement with data in S. cerevisiae, FLAGtagged endosomal syntaxin Pep12 largely localized to P13 membranes, whereas the soluble enzyme hexokinase localized to the S100 fraction.…”

Section: A Proportion Of Palb Associates With Membranes-supporting

confidence: 63%

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Physiological Involvement in pH Signaling of Vps24-mediated Recruitment of Aspergillus PalB Cysteine Protease to ESCRT-III

Rodríguez-Galán

Galindo

Hervás-Aguilar

et al. 2009

Journal of Biological Chemistry

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Section: A Proportion Of Palb Associates With Membranes-supporting

confidence: 63%

“…Subcellular Fractionation Experiments-These were carried out at 4°C following a S. cerevisiae protocol (48,49). Mycelia cultured in minimal medium (50) were protoplasted with Glucanex (Novo CH-4243).…”

Section: Methodsmentioning

confidence: 99%

Physiological Involvement in pH Signaling of Vps24-mediated Recruitment of Aspergillus PalB Cysteine Protease to ESCRT-III

Rodríguez-Galán

Galindo

Hervás-Aguilar

et al. 2009

Journal of Biological Chemistry

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“…To biochemically confirm the organellar distribution of Ntg1 and Ntg2, sucrose gradient subcellular fractionation (see Materials and Methods) was performed on lysates from cells expressing Ntg1-TAP or Ntg2-TAP to separate nuclear and mitochondrial fractions. Nuclear and mitochondrial protein lysate fractions were evaluated for purity by use of antibodies against a nuclear protein, Nop1, and a mitochondrial membrane protein, Por1 (62). Mitochondrial fractions were free of nuclear proteins, as indicated by the detection of Por1 but not Nop1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Anti-TAP, anti-Por1 (1:25,000 dilution; MitoSciences), and anti-Nop1 (1:25,000 dilution; EnCor) antibodies were employed for Western analysis. Anti-Nop1 and anti-Por1 antibodies were used to ensure enrichment of nuclear (Nop1) and mitochondrial (Por1) fractions (62). To optimize visualization, Western blot exposures were variable for each protein analyzed.…”

Section: Methodsmentioning

confidence: 99%

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

Griffiths

Swartzlander

Meadows³

et al. 2009

Molecular and Cellular Biology

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DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.Oxidative DNA damage, which occurs frequently in all cells, is linked to aging and human disease, such as cancer and various degenerative disorders (6,13,45,81,83). Reactive oxygen species (ROS) are a by-product of normal cellular metabolic processes that can cause oxidative damage to DNA, lipids, and proteins (79). Unrepaired oxidative DNA lesions can result in mutations and lead to arrest of both DNA replication and transcription (34). In order to combat such continuous insults to the genome, cells have evolved DNA repair and DNA damage tolerance pathways (2).Base excision repair (BER) is the primary process by which oxidative DNA damage is repaired (74,90). BER is initiated by the recognition and excision of a base lesion by an N-glycosylase, resulting in an apurinic/apyrimidinic (AP) site (47, 48). The resulting AP site is processed by an AP endonuclease or an AP lyase, which cleaves the sugar-phosphate DNA backbone on the 5Ј side or 3Ј side of the AP site, respectively (5). Subsequent processing involving DNA repair polymerases replaces the excised nucleotides, and DNA ligase completes the repair process (8).Very little is known about how eukaryotic cells regulate events that initiate BER in response to oxidative stress. Deleterious oxidative DNA damage can occur in both nuclear and mitochondrial genomes, adding a level of complexity to this cellular response. In this case, the intracellular localization of BER proteins would be regulated dynamically in response to the introduction of either nuclear or mitochondrial DNA damage. Controlled protein localization has been implicate...

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“…Rgd1p was tagged with the 6ϫ HA epitope at its C terminus, and the corresponding gene was integrated at its own locus in the control strain. The harvested cells were lysed, and after clarification of the lysate at 500 ϫ g, the supernatant was centrifuged at 13,000 ϫ g. The P13 pellet contains the plasma membrane, vacuole, mitochondria, ER, and cis-Golgi network; secretory vesicles and late Golgi vesicles are present in the supernatant (10,59). …”

Section: Figmentioning

confidence: 99%

Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

et al. 2012

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e Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

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Overview of Subcellular Fractionation Procedures for the YeastSaccharomyces cerevisiae

Cited by 15 publications

References 57 publications

Physiological Involvement in pH Signaling of Vps24-mediated Recruitment of Aspergillus PalB Cysteine Protease to ESCRT-III

Physiological Involvement in pH Signaling of Vps24-mediated Recruitment of Aspergillus PalB Cysteine Protease to ESCRT-III

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

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