Ll fraction of Pab1, eIF4G1 and eIF4G2 associates with Gis2-GFP (Figure 1B, lane 4). Treatment of the lysates with RNase A revealed that the interaction of Gis2-GFP with all three proteins depends on RNA (Figure 1B, lanes 5?). Although the amounts of Pab1, eIF4G1 and eIF4G2 associated with Gis2-GFP was low, these proteins were not detected in an immunoprecipitate from an untagged strain (lane 3). Additionally, reprobing of the blot with antibodies to 3-phosphoglycerate kinase, which is among the most abundant S. cerevisiae proteins [34], failed to detect this protein in immunoprecipitates (Figure 1B). Finally, although the Ded1 ATPase, which interacts with eIF4G [35], was an abundant component of the Gis2-TAP eluate (Table S1), we also failed to detect this protein in our immunoprecipitates (data not shown), consistent with reports that it is a frequent 34540-22-2 site contaminant of tandem affinity purifications [29]. We confirmed the interactions between Gis2, Pab1 and eIF4G by using anti-GFP antibodies to immunoprecipitate from strains that carried Pab1-GFP, eIF4G1-GFP or eIF4G2-GFP and also contained Gis2 fused to 3 copies of FLAG. Western blotting with anti-FLAG revealed Gis2-(FLAG)3 in both the Pab1-GFP and eIF4G2-GFP immunoprecipitates (Figure 1C), but did not detect this protein in the eIF4G1-GFP immunoprecipitate. We conclude that a small fraction of Gis2 associates with both Pab1 and eIF4G2 and possibly also eIF4G1, and that this interaction requires RNA.Gis2 Accumulates in P-bodies and Stress Granules during Glucose Deprivation and Growth in Stationary PhasePab1, eIF4G1 and eIF4G2 are all components of stress granules (also called EGP bodies), cytoplasmic mRNA-containing granules that form when translation initiation is impaired [37?0]. We therefore examined whether Gis2 was a component of stress granules 18325633 or related structures called processing bodies (P-bodies) that share some components with stress granules, but also contain components of the mRNA decapping and 5′ to 3′ decay machinery [39,41]. Strains in which the chromosomal GIS2 was fused to mCherry (mCh) were used, together with P-body and stress granule markers fused to GFP, to localize Gis2 following order H 4065 stresses that cause accumulation of these RNP granules. First, we examined the effects of glucose deprivation on Gis2 localization. Both P-bodies and stress granules become prominent when yeast cells are shifted to media lacking glucose for 10?30 min [37,38,42]. Although Gis2-mCh showed homogeneous cytoplasmic staining during logarithmic growth in rich media, a fraction localized to discrete cytoplasmic granules following 10 min of glucose deprivation (Figure 3A). Examination of two P-body markers, Dcp2, a subunit of the mRNA decapping enzyme, and Edc3, an enhancer of decapping [43], revealed that most Gis2 foci localized 1527786 with GFP-tagged forms of these proteins (Figure 3A). However, as only 57 of the Dcp2-GFP foci and 25 of the Edc3-GFP foci co-localized with Gis2-mCh, many Pbodies do not contain Gis2-mCh. Most Gis2-mCh foci also colocalized with the stress granule markers Pab1-GFP, eIF4G1-GFP, eIF4G2-GFP and Pub1-GFP (Figure 3B), consistent with reports that stress granule and P-body markers are often found in the same foci in budding yeast [37,44]. Notably, most Pab1-GFP (75 ), eIF4G1-GFP (87 ) and eIF4G2-GFP (65 ) foci also containedGis2 and CNBP Are Components of RNP GranulesFigure 1. Gis2 associates with proteins involved in translation initiation. (A) After tandem affinity purifi.Ll fraction of Pab1, eIF4G1 and eIF4G2 associates with Gis2-GFP (Figure 1B, lane 4). Treatment of the lysates with RNase A revealed that the interaction of Gis2-GFP with all three proteins depends on RNA (Figure 1B, lanes 5?). Although the amounts of Pab1, eIF4G1 and eIF4G2 associated with Gis2-GFP was low, these proteins were not detected in an immunoprecipitate from an untagged strain (lane 3). Additionally, reprobing of the blot with antibodies to 3-phosphoglycerate kinase, which is among the most abundant S. cerevisiae proteins [34], failed to detect this protein in immunoprecipitates (Figure 1B). Finally, although the Ded1 ATPase, which interacts with eIF4G [35], was an abundant component of the Gis2-TAP eluate (Table S1), we also failed to detect this protein in our immunoprecipitates (data not shown), consistent with reports that it is a frequent contaminant of tandem affinity purifications [29]. We confirmed the interactions between Gis2, Pab1 and eIF4G by using anti-GFP antibodies to immunoprecipitate from strains that carried Pab1-GFP, eIF4G1-GFP or eIF4G2-GFP and also contained Gis2 fused to 3 copies of FLAG. Western blotting with anti-FLAG revealed Gis2-(FLAG)3 in both the Pab1-GFP and eIF4G2-GFP immunoprecipitates (Figure 1C), but did not detect this protein in the eIF4G1-GFP immunoprecipitate. We conclude that a small fraction of Gis2 associates with both Pab1 and eIF4G2 and possibly also eIF4G1, and that this interaction requires RNA.Gis2 Accumulates in P-bodies and Stress Granules during Glucose Deprivation and Growth in Stationary PhasePab1, eIF4G1 and eIF4G2 are all components of stress granules (also called EGP bodies), cytoplasmic mRNA-containing granules that form when translation initiation is impaired [37?0]. We therefore examined whether Gis2 was a component of stress granules 18325633 or related structures called processing bodies (P-bodies) that share some components with stress granules, but also contain components of the mRNA decapping and 5′ to 3′ decay machinery [39,41]. Strains in which the chromosomal GIS2 was fused to mCherry (mCh) were used, together with P-body and stress granule markers fused to GFP, to localize Gis2 following stresses that cause accumulation of these RNP granules. First, we examined the effects of glucose deprivation on Gis2 localization. Both P-bodies and stress granules become prominent when yeast cells are shifted to media lacking glucose for 10?30 min [37,38,42]. Although Gis2-mCh showed homogeneous cytoplasmic staining during logarithmic growth in rich media, a fraction localized to discrete cytoplasmic granules following 10 min of glucose deprivation (Figure 3A). Examination of two P-body markers, Dcp2, a subunit of the mRNA decapping enzyme, and Edc3, an enhancer of decapping [43], revealed that most Gis2 foci localized 1527786 with GFP-tagged forms of these proteins (Figure 3A). However, as only 57 of the Dcp2-GFP foci and 25 of the Edc3-GFP foci co-localized with Gis2-mCh, many Pbodies do not contain Gis2-mCh. Most Gis2-mCh foci also colocalized with the stress granule markers Pab1-GFP, eIF4G1-GFP, eIF4G2-GFP and Pub1-GFP (Figure 3B), consistent with reports that stress granule and P-body markers are often found in the same foci in budding yeast [37,44]. Notably, most Pab1-GFP (75 ), eIF4G1-GFP (87 ) and eIF4G2-GFP (65 ) foci also containedGis2 and CNBP Are Components of RNP GranulesFigure 1. Gis2 associates with proteins involved in translation initiation. (A) After tandem affinity purifi.
