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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

Ydj1 is necessary for the formation of PB foci. (A) Fluorescence microscopic images of Dhh1−GFP, Lsm1−GFP, or Edc3−GFP in wild-type (YDJ1) or mutant (ydj1∆) cells after a 30-min glucose depletion, or overnight culture. Scale bar = 10 µm. (B) Microscopic images of Dhh1−GFP in ydj1∆ mutants transformed with centromere-containing plasmids harboring the corresponding genes. All strains were induced to form foci by 30 min of glucose depletion. Scale bar = 10 µm. (C) Quantitation of the percent of cells with at least 2 Dhh1−GFP foci after 30 min of glucose depletion for the strains in (B). Data presented are the average of at least two replicate experiments in which a minimum of 40 cells were counted. Error bars represent the standard deviation of the replicate measurements. (*only 1 replicate quantified for Hsp104.)
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fig3: Ydj1 is necessary for the formation of PB foci. (A) Fluorescence microscopic images of Dhh1−GFP, Lsm1−GFP, or Edc3−GFP in wild-type (YDJ1) or mutant (ydj1∆) cells after a 30-min glucose depletion, or overnight culture. Scale bar = 10 µm. (B) Microscopic images of Dhh1−GFP in ydj1∆ mutants transformed with centromere-containing plasmids harboring the corresponding genes. All strains were induced to form foci by 30 min of glucose depletion. Scale bar = 10 µm. (C) Quantitation of the percent of cells with at least 2 Dhh1−GFP foci after 30 min of glucose depletion for the strains in (B). Data presented are the average of at least two replicate experiments in which a minimum of 40 cells were counted. Error bars represent the standard deviation of the replicate measurements. (*only 1 replicate quantified for Hsp104.)

Mentions: The Hsp40 family protein Ydj1 has an LC domain (Table S7), binds Q/N-prion domain proteins (Summers et al. 2009), and in our proteomic studies associates with Dhh1−GFP only in the glucose depleted condition (Table 2 and Table S5). To test the effects of Ydj1 on PB assembly, we examined the ability of Dhh1–GFP, Lsm1-GFP or Edc3-GFP to localize to cytoplasmic foci in a ydj1∆ mutant background. In contrast to wild-type cells, Dhh1–GFP or Lsm1-GFP foci formation are defective in ydj1∆ mutant cells that are grown in glucose-depleted media (Figure 3A). The formation of Dhh1–GFP or Lsm1-GFP foci when cells were grown to saturation, another PB inducing condition, also was drastically reduced in the ydj1∆ mutant background. In contrast, the loss of YDJ1 had minimal effects on Edc3-GFP aggregation (Figure 3A). We also tested whether deletion of other members of the Hsp70, Hsp90 and Hsp110 families, including ssa1∆, ssa2∆, hsc82∆, hsp82∆, or hsp104∆, affected the stress induction of Dhh1–GFP foci and found that Dhh1–GFP foci formation to be the same as wild-type in all these mutant backgrounds (Figure S3). The Dhh1−GFP foci assembly defect in ydj1∆ mutant (YAD557) was complemented by plasmids containing a wild-type copy of YDJ1, but not any of the other related protein chaperones tested (Figure 3, B and C). We also examined the effects of the ydj1∆ mutation on protein levels of Dhh1–GFP, Lsm1-GFP or Edc3-GFP, and found that in all three cases the protein levels are decreased to about 20% of the levels in wild-type cells (Figure S4A). Reintroducing the wild-type YDJ1 did not restore Dhh1–GFP level to that of wild-type (Figure S4B). This result suggests that the interplay between YDJ1, protein levels, and RNA granule assembly is complex and not fully resolved by these experiments. The interpretation of these data may be confounded by the differences between the expression of YDJ1 from the plasmid vs. the endogenous chromosomal locus. Furthermore, there may be a residual epigenetic effect of the deletion of ydj1 that has not been addressed by the complementation; Ydj1 is known to be central in prion formation and propagation (Summers et al. 2009). It is also possible that when wild-type YDJ1 is introduced, the levels of Lsm1 or Edc3 in the ydj1∆ strain that expresses Dhh1–GFP are restored enough to drive Dhh1–GFP foci assembly. Taken together, these results suggest that Ydj1 is required for the formation of Dhh1- and Lsm1-containing cytoplasmic foci in response to glucose limitation stress.


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)

Ydj1 is necessary for the formation of PB foci. (A) Fluorescence microscopic images of Dhh1−GFP, Lsm1−GFP, or Edc3−GFP in wild-type (YDJ1) or mutant (ydj1∆) cells after a 30-min glucose depletion, or overnight culture. Scale bar = 10 µm. (B) Microscopic images of Dhh1−GFP in ydj1∆ mutants transformed with centromere-containing plasmids harboring the corresponding genes. All strains were induced to form foci by 30 min of glucose depletion. Scale bar = 10 µm. (C) Quantitation of the percent of cells with at least 2 Dhh1−GFP foci after 30 min of glucose depletion for the strains in (B). Data presented are the average of at least two replicate experiments in which a minimum of 40 cells were counted. Error bars represent the standard deviation of the replicate measurements. (*only 1 replicate quantified for Hsp104.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Ydj1 is necessary for the formation of PB foci. (A) Fluorescence microscopic images of Dhh1−GFP, Lsm1−GFP, or Edc3−GFP in wild-type (YDJ1) or mutant (ydj1∆) cells after a 30-min glucose depletion, or overnight culture. Scale bar = 10 µm. (B) Microscopic images of Dhh1−GFP in ydj1∆ mutants transformed with centromere-containing plasmids harboring the corresponding genes. All strains were induced to form foci by 30 min of glucose depletion. Scale bar = 10 µm. (C) Quantitation of the percent of cells with at least 2 Dhh1−GFP foci after 30 min of glucose depletion for the strains in (B). Data presented are the average of at least two replicate experiments in which a minimum of 40 cells were counted. Error bars represent the standard deviation of the replicate measurements. (*only 1 replicate quantified for Hsp104.)
Mentions: The Hsp40 family protein Ydj1 has an LC domain (Table S7), binds Q/N-prion domain proteins (Summers et al. 2009), and in our proteomic studies associates with Dhh1−GFP only in the glucose depleted condition (Table 2 and Table S5). To test the effects of Ydj1 on PB assembly, we examined the ability of Dhh1–GFP, Lsm1-GFP or Edc3-GFP to localize to cytoplasmic foci in a ydj1∆ mutant background. In contrast to wild-type cells, Dhh1–GFP or Lsm1-GFP foci formation are defective in ydj1∆ mutant cells that are grown in glucose-depleted media (Figure 3A). The formation of Dhh1–GFP or Lsm1-GFP foci when cells were grown to saturation, another PB inducing condition, also was drastically reduced in the ydj1∆ mutant background. In contrast, the loss of YDJ1 had minimal effects on Edc3-GFP aggregation (Figure 3A). We also tested whether deletion of other members of the Hsp70, Hsp90 and Hsp110 families, including ssa1∆, ssa2∆, hsc82∆, hsp82∆, or hsp104∆, affected the stress induction of Dhh1–GFP foci and found that Dhh1–GFP foci formation to be the same as wild-type in all these mutant backgrounds (Figure S3). The Dhh1−GFP foci assembly defect in ydj1∆ mutant (YAD557) was complemented by plasmids containing a wild-type copy of YDJ1, but not any of the other related protein chaperones tested (Figure 3, B and C). We also examined the effects of the ydj1∆ mutation on protein levels of Dhh1–GFP, Lsm1-GFP or Edc3-GFP, and found that in all three cases the protein levels are decreased to about 20% of the levels in wild-type cells (Figure S4A). Reintroducing the wild-type YDJ1 did not restore Dhh1–GFP level to that of wild-type (Figure S4B). This result suggests that the interplay between YDJ1, protein levels, and RNA granule assembly is complex and not fully resolved by these experiments. The interpretation of these data may be confounded by the differences between the expression of YDJ1 from the plasmid vs. the endogenous chromosomal locus. Furthermore, there may be a residual epigenetic effect of the deletion of ydj1 that has not been addressed by the complementation; Ydj1 is known to be central in prion formation and propagation (Summers et al. 2009). It is also possible that when wild-type YDJ1 is introduced, the levels of Lsm1 or Edc3 in the ydj1∆ strain that expresses Dhh1–GFP are restored enough to drive Dhh1–GFP foci assembly. Taken together, these results suggest that Ydj1 is required for the formation of Dhh1- and Lsm1-containing cytoplasmic foci in response to glucose limitation stress.

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