E consistent with a model in which both Gis2 and CNBP participate in mRNA handling during stress.A Small Fraction of Gis2 may Associate with PolyribosomesThe large number of proteins from the small and large ribosomal subunits in our Gis2-TAP purification (Table S1), coupled with a report that Gis2 sediments with polyribosomes [15], prompted us to examine whether Gis2 was polyribosomeassociated. GIS2-GFP lysates were prepared in the presence of cycloheximide, which stabilizes translating ribosomes, and subjected to sucrose gradient sedimentation (Figure 2A). Western blotting revealed that most Gis2-GFP sedimented at the top of the gradient (fractions 1?; 55.8 ). However, some Gis2-GFP sedimented in fractions containing ribosomal subunits and monoribosomes (fractions 4?0; 39.7 ), and a small amount was found in polyribosome-containing fractions (fractions 11?1; 4.5 ). Reprobing to detect Pab1 revealed that this protein was found throughout the gradient, as described [36,37]. To determine if the Gis2-GFP that sedimented with polyribosomes was indeed polyribosome-associated, we disrupted polyribosomes before performing gradient fractionation. Experiments in which we omitted the cycloheximide resulted in decreased polyribosomes, with a concomitant BIBS39 increase in 80S monoribosomes (Figure 2B). Treatment of the lysate with micrococcal nuclease to degrade portions of mRNA that are not protected by ribosomes also converted most polysomes to 80S monosomes (Figure 2D). Western blotting to detect the large ribosomal subunit proteins Rpl1A and Rpl1B confirmed that both treatments were effective at disrupting polyribosomes (Figures 2B and 2D). Following both treatments, the amount of Gis2-GFP present in polyribosome-containing fractions (fractions 11?1) was reduced (Figures 2B and 2D) (to 1.3 and 1.2 , respectively). 18325633 Thus, a small fraction of Gis2-GFP may be polyribosome-associated.Results Gis2 Interacts with Components Involved in mRNA TranslationTo identify Gis2-associated proteins, we subjected a strain in which Gis2 was fused to a TAP module to two rounds of affinity purification. Silver staining of the final eluate revealed Gis2 and several bands that were not detected in a parallel purification from an untagged strain (Figure 1A). Proteins in both 24195657 eluates were Fruquintinib supplier analyzed using multidimensional protein identification technology (MUDPIT) [28]. After filtering out proteins that are common contaminants of TAP purifications [29], the most abundant proteins in the Gis2-TAP eluate included the poly(A) binding protein Pab1, the two isoforms of the translation initiation factor eIF4G (eIF4G1 and eIF4G2) and numerous ribosomal proteins (Table S1). Several other proteins were also linked to translation initiation, such as the cap-binding protein eIF4E [30], or mRNA stability, such as Xrn1, the major 59 to 39 exoribonuclease that carries out mRNA decay [31]. To validate the interactions, we focused on Pab1, eIF4G1 and eIF4G2. Pab1 and eIF4G, together with eIF4E and eIF4A, are involved in cap-dependent translation initiation [32]. Specifically, eIF4G, together with eIF4E and the DExD/H helicase eIF4A, forms the cap-binding complex eIF4F. Association of eIF4G with Pab1, which binds the mRNA poly(A) tail, circularizes the mRNA and increases the efficiency of recruiting 43S initiation complexes [32,33]. Using anti-GFP antibodies to immunoprecipitate from GIS2-GFP cell lysates, followed by Western blotting of proteins in immunoprecipitates, we confirmed that a sma.E consistent with a model in which both Gis2 and CNBP participate in mRNA handling during stress.A Small Fraction of Gis2 may Associate with PolyribosomesThe large number of proteins from the small and large ribosomal subunits in our Gis2-TAP purification (Table S1), coupled with a report that Gis2 sediments with polyribosomes [15], prompted us to examine whether Gis2 was polyribosomeassociated. GIS2-GFP lysates were prepared in the presence of cycloheximide, which stabilizes translating ribosomes, and subjected to sucrose gradient sedimentation (Figure 2A). Western blotting revealed that most Gis2-GFP sedimented at the top of the gradient (fractions 1?; 55.8 ). However, some Gis2-GFP sedimented in fractions containing ribosomal subunits and monoribosomes (fractions 4?0; 39.7 ), and a small amount was found in polyribosome-containing fractions (fractions 11?1; 4.5 ). Reprobing to detect Pab1 revealed that this protein was found throughout the gradient, as described [36,37]. To determine if the Gis2-GFP that sedimented with polyribosomes was indeed polyribosome-associated, we disrupted polyribosomes before performing gradient fractionation. Experiments in which we omitted the cycloheximide resulted in decreased polyribosomes, with a concomitant increase in 80S monoribosomes (Figure 2B). Treatment of the lysate with micrococcal nuclease to degrade portions of mRNA that are not protected by ribosomes also converted most polysomes to 80S monosomes (Figure 2D). Western blotting to detect the large ribosomal subunit proteins Rpl1A and Rpl1B confirmed that both treatments were effective at disrupting polyribosomes (Figures 2B and 2D). Following both treatments, the amount of Gis2-GFP present in polyribosome-containing fractions (fractions 11?1) was reduced (Figures 2B and 2D) (to 1.3 and 1.2 , respectively). 18325633 Thus, a small fraction of Gis2-GFP may be polyribosome-associated.Results Gis2 Interacts with Components Involved in mRNA TranslationTo identify Gis2-associated proteins, we subjected a strain in which Gis2 was fused to a TAP module to two rounds of affinity purification. Silver staining of the final eluate revealed Gis2 and several bands that were not detected in a parallel purification from an untagged strain (Figure 1A). Proteins in both 24195657 eluates were analyzed using multidimensional protein identification technology (MUDPIT) [28]. After filtering out proteins that are common contaminants of TAP purifications [29], the most abundant proteins in the Gis2-TAP eluate included the poly(A) binding protein Pab1, the two isoforms of the translation initiation factor eIF4G (eIF4G1 and eIF4G2) and numerous ribosomal proteins (Table S1). Several other proteins were also linked to translation initiation, such as the cap-binding protein eIF4E [30], or mRNA stability, such as Xrn1, the major 59 to 39 exoribonuclease that carries out mRNA decay [31]. To validate the interactions, we focused on Pab1, eIF4G1 and eIF4G2. Pab1 and eIF4G, together with eIF4E and eIF4A, are involved in cap-dependent translation initiation [32]. Specifically, eIF4G, together with eIF4E and the DExD/H helicase eIF4A, forms the cap-binding complex eIF4F. Association of eIF4G with Pab1, which binds the mRNA poly(A) tail, circularizes the mRNA and increases the efficiency of recruiting 43S initiation complexes [32,33]. Using anti-GFP antibodies to immunoprecipitate from GIS2-GFP cell lysates, followed by Western blotting of proteins in immunoprecipitates, we confirmed that a sma.
