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Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly.

Cary GA, Vinh DB, May P, Kuestner R, Dudley AM - G3 (Bethesda) (2015)

Bottom Line: Many of these proteins have been shown to form foci in response to other stresses.Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins.Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, Washington 98109 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

No MeSH data available.


Related in: MedlinePlus

Immunoisolation of Dhh1−GFP. (A) Fluorescence microscopy showing the aggregation of Dhh1−GFP into cytoplasmic foci in cells grown in media with and without glucose for 30 min. Scale bar = 10 µm. (B) Silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of immunoprecipitation fractions of Dhh1−GFP isolated from cells grown in +glucose (YPD) and −glucose (YEP) media; and from the negative control BY4741 cells grown in −glucose. The identities of some of the dominant bands [marked with an asterisk, Dhh1−GFP (red), and dominant coisolated P-bodies proteins (blue)] are based on the apparent molecular weight and by mass spectrometry from other purifications. MW, molecular weight of standard proteins.
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fig1: Immunoisolation of Dhh1−GFP. (A) Fluorescence microscopy showing the aggregation of Dhh1−GFP into cytoplasmic foci in cells grown in media with and without glucose for 30 min. Scale bar = 10 µm. (B) Silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of immunoprecipitation fractions of Dhh1−GFP isolated from cells grown in +glucose (YPD) and −glucose (YEP) media; and from the negative control BY4741 cells grown in −glucose. The identities of some of the dominant bands [marked with an asterisk, Dhh1−GFP (red), and dominant coisolated P-bodies proteins (blue)] are based on the apparent molecular weight and by mass spectrometry from other purifications. MW, molecular weight of standard proteins.

Mentions: Anti-GFP IgG (catalog no. 11814460001; Roche) coupled to magnetic Protein-G Dynabeads (Invitrogen) was used to capture Dhh1−GFP protein complexes from yeast cell lysate. Anti-GFP was first crosslinked to protein-G bead in 20 mM dimethyl pimelimidate (Thermo Scientific) with the use of protocols recommended by the manufacturer. A ratio of 30 mg of Ab-proG beads to 300 mg of total protein captured >90% of Dhh1−GFP in the cell lysate, as determined by Western blot analysis. Cell lysate generated from 4 L of cell culture was used to obtain sufficient material for MS. To generate a lysate supernatant, cell powder grindate was thawed quickly by resuspending in 1.5 volumes of RB buffer [30 mM K-Hepes (pH 7.4), 150 mM KCl, 2 mM MgCl2, 0.2% NP-40, 0.1% Tween-20, 1 mM dithiothreitol, yeast protease inhibitor cocktail (Sigma-Aldrich), and RNase-inhibitor (Ambion / Millipore)]. The suspension was subsequently cleared by low-speed centrifugation (3000g) for 5−7 min at 4°. The resulting supernatant was incubated with anti-GFP-protG beads for 30 min at 4° with gentle mixing. The beads subsequently were separated from the supernatant and washed extensively with RB buffer containing progressively less detergents (0.1% NP-40/0.05% Tween-20). After the final wash, the beads were divided into separate fractions for elution of protein (88% of total) and RNA (12% of total). Protein was eluted in a solution of 0.1% sodium dodecyl sulfate (SDS), 30 mM Hepes (pH 7.4), protease, and RNase inhibitors for 30 min at room temperature. A fraction of the eluted proteins was analyzed immediately by Silver staining (Pierce/ThermoScientific) and Western blot (Licor Odyssey) with the use of a different anti-GFP antibody (catalog no. 632381; Clontech), while the remaining was flash frozen in liquid nitrogen and stored at −80°. The identities of some of the dominant bands on silver stained SDS gels (Figure 1, marked with asterisks) are PB components assigned by their apparent molecular weight by gel migration and MS from other purifications. For RNA isolation, beads were incubated with 1% SDS, 30 mM Hepes (pH 7.4), protease and RNase inhibitors for 30 min at room temperature. The resulting supernatant was mixed with an equal volume of TES buffer [1% SDS in 10 mM Tris (pH 7.5), 1 mM ethylenediaminetetraacetic acid], added to a total equal volume of acid phenol (pH 4), and incubated at 65° for 60 min. Samples were spun at 15,000 g for 5 min. Then, the aqueous phase was collected and extracted again with phenol:chloroform, followed by precipitation with cold ethanol. Final pellets were resuspended in TE and stored at −80°.


Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly.

Cary GA, Vinh DB, May P, Kuestner R, Dudley AM - G3 (Bethesda) (2015)

Immunoisolation of Dhh1−GFP. (A) Fluorescence microscopy showing the aggregation of Dhh1−GFP into cytoplasmic foci in cells grown in media with and without glucose for 30 min. Scale bar = 10 µm. (B) Silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of immunoprecipitation fractions of Dhh1−GFP isolated from cells grown in +glucose (YPD) and −glucose (YEP) media; and from the negative control BY4741 cells grown in −glucose. The identities of some of the dominant bands [marked with an asterisk, Dhh1−GFP (red), and dominant coisolated P-bodies proteins (blue)] are based on the apparent molecular weight and by mass spectrometry from other purifications. MW, molecular weight of standard proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4632068&req=5

fig1: Immunoisolation of Dhh1−GFP. (A) Fluorescence microscopy showing the aggregation of Dhh1−GFP into cytoplasmic foci in cells grown in media with and without glucose for 30 min. Scale bar = 10 µm. (B) Silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of immunoprecipitation fractions of Dhh1−GFP isolated from cells grown in +glucose (YPD) and −glucose (YEP) media; and from the negative control BY4741 cells grown in −glucose. The identities of some of the dominant bands [marked with an asterisk, Dhh1−GFP (red), and dominant coisolated P-bodies proteins (blue)] are based on the apparent molecular weight and by mass spectrometry from other purifications. MW, molecular weight of standard proteins.
Mentions: Anti-GFP IgG (catalog no. 11814460001; Roche) coupled to magnetic Protein-G Dynabeads (Invitrogen) was used to capture Dhh1−GFP protein complexes from yeast cell lysate. Anti-GFP was first crosslinked to protein-G bead in 20 mM dimethyl pimelimidate (Thermo Scientific) with the use of protocols recommended by the manufacturer. A ratio of 30 mg of Ab-proG beads to 300 mg of total protein captured >90% of Dhh1−GFP in the cell lysate, as determined by Western blot analysis. Cell lysate generated from 4 L of cell culture was used to obtain sufficient material for MS. To generate a lysate supernatant, cell powder grindate was thawed quickly by resuspending in 1.5 volumes of RB buffer [30 mM K-Hepes (pH 7.4), 150 mM KCl, 2 mM MgCl2, 0.2% NP-40, 0.1% Tween-20, 1 mM dithiothreitol, yeast protease inhibitor cocktail (Sigma-Aldrich), and RNase-inhibitor (Ambion / Millipore)]. The suspension was subsequently cleared by low-speed centrifugation (3000g) for 5−7 min at 4°. The resulting supernatant was incubated with anti-GFP-protG beads for 30 min at 4° with gentle mixing. The beads subsequently were separated from the supernatant and washed extensively with RB buffer containing progressively less detergents (0.1% NP-40/0.05% Tween-20). After the final wash, the beads were divided into separate fractions for elution of protein (88% of total) and RNA (12% of total). Protein was eluted in a solution of 0.1% sodium dodecyl sulfate (SDS), 30 mM Hepes (pH 7.4), protease, and RNase inhibitors for 30 min at room temperature. A fraction of the eluted proteins was analyzed immediately by Silver staining (Pierce/ThermoScientific) and Western blot (Licor Odyssey) with the use of a different anti-GFP antibody (catalog no. 632381; Clontech), while the remaining was flash frozen in liquid nitrogen and stored at −80°. The identities of some of the dominant bands on silver stained SDS gels (Figure 1, marked with asterisks) are PB components assigned by their apparent molecular weight by gel migration and MS from other purifications. For RNA isolation, beads were incubated with 1% SDS, 30 mM Hepes (pH 7.4), protease and RNase inhibitors for 30 min at room temperature. The resulting supernatant was mixed with an equal volume of TES buffer [1% SDS in 10 mM Tris (pH 7.5), 1 mM ethylenediaminetetraacetic acid], added to a total equal volume of acid phenol (pH 4), and incubated at 65° for 60 min. Samples were spun at 15,000 g for 5 min. Then, the aqueous phase was collected and extracted again with phenol:chloroform, followed by precipitation with cold ethanol. Final pellets were resuspended in TE and stored at −80°.

Bottom Line: Many of these proteins have been shown to form foci in response to other stresses.Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins.Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, Washington 98109 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

No MeSH data available.


Related in: MedlinePlus