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, 21 (12), 1922-36

O-GlcNAc Cycling Enzymes Associate With the Translational Machinery and Modify Core Ribosomal Proteins

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O-GlcNAc Cycling Enzymes Associate With the Translational Machinery and Modify Core Ribosomal Proteins

Quira Zeidan et al. Mol Biol Cell.

Abstract

Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.

Figures

Figure 1.
Figure 1.
Preparations enriched in ribosomal components contain numerous O-GlcNAc proteins. (A) Postmitochondrial (PM), postribosomal (PR), and ribosomal (Ribo) preparations were obtained by subcellular fractionation of rat pancreas and subjected to galactosyltransferase labeling in the presence of UDP-[3H]Gal. Top, autoradiographs of the gels stained with G250 seen in the bottom panels. Many O-GlcNAcylated low-molecular-weight proteins (<50 kDa) are observed in the Ribo preparation (arrowheads). No substrate (NS) and positive ovalbumin (Ovb) controls are included. Reactions containing all the components except galactosyltransferase (−GalT) are shown in the top right panel. (B) Ribosomal preparations from rat liver were obtained and labeled as in A. Partial pretreatment with commercial hexosaminidase (Ribo+Hex) decreases labeling by galactosyltransferase. (C) Fractions from polysome profiles (top panel) obtained from HepG2 cells growing under normal conditions were TCA precipitated, separated by electrophoresis, and subjected to immunoblot (IB) analysis with O-GlcNAc antibody (middle). A competition control obtained by pre-incubating the antibody with 1 M GlcNAc shows specificity of the labeling (bottom). Sedimentation was from left to right. Data shown are representative of at least three independent experiments.
Figure 2.
Figure 2.
Extensive separation of ribosomes allows the detection of multiple individual O-GlcNAcylated proteins. (A) Strategy for separation and identification of ribosomal proteins modified with O-GlcNAc. (B) Representative chromatogram obtained after HPLC separation of a purified rat liver ribosomal fraction over a reverse-phase C8 column. (C) Reverse-phase HPLC fractions containing ribosomal protein peaks from HeLa cells were separated by one-dimensional gel electrophoresis and subjected to lectin blot (LB) with sWGA (top panels). Fractions were loaded in subsequent order of retention time (early to late from left to right). Comparison with the corresponding G250-stained gel (bottom panels) shows that many, but not all, proteins present in the fractions contain O-GlcNAc. Two examples (asterisk and arrowhead) show that the lectin signal for O-GlcNAc has different stoichiometries when compared with equivalent levels of total protein. (D) Fractions as in C were subjected to galactosyltransferase labeling in the presence of UDP-[3H]Gal (top panels). Bottom panels show G250 protein stain of the top panel. No substrate (N), total preparation (previous to RP-HPLC) with galactosyltransferase (+), and total preparation without galactosyltransferase (−) controls are included. Data shown are representative of at least three independent experiments.
Figure 3.
Figure 3.
Identification of ribosomal proteins modified with O-GlcNAc. (A) Reverse-phase HPLC fractions containing ribosomal protein peaks from HeLa cells were separated by one-dimensional gel electrophoresis and subjected to immunoblot (IB) analysis with O-GlcNAc-specific antibody (top panels). Fractions were loaded in subsequent order of retention time (early to late from left to right). Corresponding G250-stained gels are shown (bottom panels). Numbered arrowheads show protein bands selected for identification by MS/MS after careful comparison and gel line-up. Identified species are shown in Table 1. (B) Same as in A but with rat liver ribosomes. Data shown are representative of >5 independent experiments.
Figure 4.
Figure 4.
MS/MS of the enriched and derivatized peptides identified the sites of O-GlcNAc modification on four rat liver ribosomal proteins. (A) Serine 72 of RPL32. (B) Serine 91 of RPL36. (C) Serine 265 of RPL6. The m/z values of the y(5) and y(6) species are 599.48 and 822.5, respectively. (D) Serine 66 of RPL29.
Figure 4.
Figure 4.
MS/MS of the enriched and derivatized peptides identified the sites of O-GlcNAc modification on four rat liver ribosomal proteins. (A) Serine 72 of RPL32. (B) Serine 91 of RPL36. (C) Serine 265 of RPL6. The m/z values of the y(5) and y(6) species are 599.48 and 822.5, respectively. (D) Serine 66 of RPL29.
Figure 5.
Figure 5.
O-GlcNAcylation of RPS6 exhibits different dynamics than nutrient-induced phosphorylation. (A) Reverse-phase HPLC fractions containing rat liver RPS6 visible by G250 and identified by MS/MS were immunoblotted (IB) for O-GlcNAc and S6. (B) Lysates from HEK293 cells transfected with HA, HA-S6, HA-S6-S135/236A, or HA-S6-S235/236D were immunoprecipitated (IP) for HA and immunoblotted (IB) for HA and O-GlcNAc. (C) Lysates from Neuro-2a cells transfected with HA or HA-S6 were glucose-deprived for the indicated times, immunoprecipitated (IP) for HA, and immunoblotted (IB) for O-GlcNAc, HA, phospho-S6 (Ser240/244), and actin. Inputs are shown in the left panel. (D) HEK293 cells transfected with HA or HA-S6 were serum-starved overnight and stimulated with serum (10%) over the indicated times. Lysates were immunoprecipitated (IP) for HA and immunblotted (IB) for HA, O-GlcNAc, and phospho-S6 (Ser240/244). The vector-only control (HA) corresponding to 60-min serum stimulation is shown. Data shown are representative of at least three independent experiments.
Figure 6.
Figure 6.
O-GlcNAc cycling enzymes associate with ribosomes. (A) Total ribosome preparation from rat liver was incubated with increasing concentrations of salt (KCl) as indicated and subjected to an additional round of purification through a sucrose cushion. Ribosomal proteins were extracted with acetic acid, acetone precipitated, separated by electrophoresis, and immunoblotted (IB) for OGT, OGAse, and S6 (top left panel and bottom panel). Densitometry analysis from four independent experiments shows the ratio of ribosome-associated OGT to RPS6 (top right panel), showing a significant difference between the 0.1 and the 0.5 M samples (P = 0.07). Bars in graph represent the values; error bars, SE. NW, no wash; DU, densitometry units; PR, postribosomal fraction. (B) Polysomal fractions from HepG2 cells growing under normal conditions were TCA precipitated, separated by electrophoresis, and subjected to immunoblot (IB) analysis with OGT, OGAse, and S6 antibodies. (SP) subpolysomal fractions. Data shown are representative of at least three independent experiments.
Figure 7.
Figure 7.
OGAse is present in purified nucleoli. (A) HeLa cells were fractionated into different subcellular components in order to obtain intact nuclei and nucleoli. Equivalent amount of total proteins from the different fractions was separated by electrophoresis and immunoblotted (IB) for tubulin, fibrillarin, OGAse, and OGT (left). Densitometry analysis from four independent experiments shows the amount of OGAse per microgram of protein loaded in every fraction (right). Bars in graph represent the values; error bars, SE. (B) Nucleoli from A were spotted onto coverslips, fixed, permeabilized, and stained for immunofluorescence confocal microscopy with OGAse (red), RPS6 (green), OGT (red), and fibrillarin (green) antibodies. Isotype (red) and secondary (red) controls are included for the OGAse antibody. Magnifications are shown in parenthesis. All pictures in each panel were exposed for equal times and subjected to the same brightness/contrast adjustments. Data shown are representative of at least three independent experiments.
Figure 8.
Figure 8.
Nucleolar stress disrupts staining of OGAse in the nucleus of cells. (A) HeLa cells were grown on coverslips, fixed, permeabilized, and stained for immunofluorescence confocal microscopy with OGAse (red) antibody. Nuclear OGAse staining shows a gradient from nucleoplasm to nucleoli (white arrow). Isotype (red) and secondary (red, tubulin shown in green) controls are included. Magnification, ×100. All pictures were exposed for equal times (50 ms) and subjected to the same brightness/contrast adjustments. (B) HeLa cells were subjected to treatments as indicated, processed as in A, and double-stained with OGAse (red) and fibrillarin (green) or OGT (red) and fibrillarin (green) antibodies. OGAse staining in the nucleus follows the same pattern as disrupted fibrillarin (white arrows). Magnifications, ×63 (OGT) and ×100 (OGAse). All pictures in each panel were exposed for equal times and subjected to the same brightness/contrast adjustments. Data shown are representative of at least three independent experiments.
Figure 9.
Figure 9.
Adenoviral-mediated overexpression of OGT causes accumulation of 60S subunits and 80S monosomes. (A) HepG2 cells were infected with adenovirus overexpressing either GFP control, OGT, or OGAse. Lysates were obtained after 48 h and immunoblotted (IB) for O-GlcNAc, OGT, OGAse, GFP, and actin to determine efficiency of overexpression. (B) HepG2 cells were infected as in A and polysome profiles were obtained 48 h after infection. Cells overexpressing OGT (vOGT) show an increase in the 60S (arrowhead) and the 80S (arrow) peaks as compared with vOGAse and vGFP control. Polysomal fractions were TCA precipitated, separated by electrophoresis, and subjected to immunoblot analysis with OGT, OGAse, and S6 antibodies. Data shown are representative of three independent experiments.

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