Journal: The Journal of Biological Chemistry

Article Title: N -Glycans on EGF domain-specific O -GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

doi: 10.1074/jbc.RA119.012280

Figure Lengend Snippet: EOGT is modified with oligomannose N-glycans. A, lectin blot analysis of FLAG-EOGT isoforms. HEK293T cells were transfected to express each FLAG-EOGT isoform. The cell lysates were subjected to immunoprecipitation with FLAG-antibody followed by detection by biotinylated ConA lectin (ConA-biotin) or EOGT-CT antibody. An asterisk indicates nonspecific bands. B, LC–MS/MS spectra of glycopeptides modified with HexNAc2Hex9 N-glycan at N263 (top) and HexNAc2Hex7 N-glycan at Asn-354 (bottom) of FLAG-EOGT. Chymotryptic or tryptic glycopeptides prepared from recombinant FLAG-EOGT were analyzed by LC–MS/MS. Fragments ions corresponding to b and y ions, peptides with truncated glycans, and glycans are shown by arrows. Blue square, HexNAc (presumably GlcNAc); green circle, hexose (presumably mannose). C, bar graphs showing relative abundance of different N-glycan glycoforms at EOGT Asn-263 and Asn-354. D, endogenous EOGT sensitivity to Endo H digestion. HEK293T cell lysates were incubated in the absence or presence of Endo H and analyzed by immunoblotting with EOGT-specific AER61 antibody. Recombinant FLAG-EOGT and FLAG-EOGTN263Q/N354Q were analyzed in parallel as controls.

Article Snippet: Next, the beads were washed extensively with the lysis buffer and eluted with 50 µl of 3× FLAG peptides (1 µg/µl, MBL) in 10 m m HEPES, pH 7.0.

Techniques: Modification, Transfection, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: N -Glycans on EGF domain-specific O -GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

doi: 10.1074/jbc.RA119.012280

Figure Lengend Snippet: Reduced O-GlcNAc stoichiometry on Notch1 in HEK293T cells expressing N-glycan-deficient EOGT. A, MS analysis of tryptic glycopeptides prepared from FLAG-Notch1-TM harboring O-GlcNAcylation sites. FLAG-Notch1-TM was expressed in EOGT-deficient HEK293T cells exogenously expressing WT or the N263Q/N354Q EOGT. EICs show the relative signal intensity corresponding to the peptides without modification (orange) or with O-GlcNAc (blue) on EGF2, EGF10, EGF11, EGF21, and EGF23 of Notch1. Note that no elongated O-GlcNAc glycoforms were detected. Data are presented as mean ± S.D. (n = 2). LC–MS/MS spectra of tryptic glycopeptides are shown in Fig. S3. B, quantification of O-GlcNAc glycans on representative EGF domains. EIC peak heights were measured and expressed as percent area. The color code is same as described in A.

Article Snippet: Next, the beads were washed extensively with the lysis buffer and eluted with 50 µl of 3× FLAG peptides (1 µg/µl, MBL) in 10 m m HEPES, pH 7.0.

Techniques: Expressing, Modification, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts

doi: 10.3390/ijms222312639

Figure Lengend Snippet: SOT1 directly interacts with CAF1 and CAF2. ( a ) Yeast two-hybrid assays indicate that SOT1 interacts with CAF1 and CAF2. SOT1 was fused to the BD vector; CAF1 (or CAF2, CRS2, and CFM2) was fused to the AD vector. The ability to grow on SD/-Trp-Leu-His-Ade dropout plates indicates an interaction between the two proteins. ( b ) Luciferase complementation assay for interactions between SOT1 and CAF1 versus CAF2. The indicated NLuc and CLuc constructs were transiently coexpressed in Nicotiana benthamiana plants through Agrobacterium-mediated transformation, and the luciferase (Luc) activity was measured after 48 h. Error bars indicate SD ( n = 4). ( c ) Coimmunoprecipitation assays showing the interaction of SOT1 with CAF1 or CAF2. 3 × HA-tagged SOT1 and 3 × FLAG-tagged CAF1 (or CAF2) were coexpressed in Arabidopsis protoplasts. Protein complexes were immunoprecipitated (IP) using an α-FLAG antibody. Immunoblot (IB) analysis of the protein presence in the immunoprecipitates using the α-FLAG and α-HA antibodies, respectively. The assay of interaction between SOT1 and Ycf4 was used as a negative control.

Article Snippet: The bound protein was eluted with 60 μL of 0.5 mg mL –1 3 × FLAG peptide (MBL, Minato-Ku, Japan) for 1 h. After elution, the proteins were separated using SDS-PAGE and detected using an anti-HA and anti-FLAG immunoblot.

Techniques: Plasmid Preparation, Luciferase, Construct, Transformation Assay, Activity Assay, Immunoprecipitation, Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts

doi: 10.3390/ijms222312639

Figure Lengend Snippet: CAF1 and CAF2 promote the binding of SOT1 to the 5′ end of ndhA transcripts. ( a ) Electrophoretic mobility shift assay (EMSA) showing the positive effect of CAF1 and CAF2 on the binding of SOT1 to the 5′ end of ndhA transcripts. The predicted binding sequence (UGGCUGAUAUUA) of SOT1 on ndhA transcripts was synthesized and labeled with biotin. Increasing concentrations of recombinant CAF1 or CAF2 proteins were incubated with 10 nM biotin-labeled probe and the MBP-SOT1 protein. The competitor used in the competition experiments was an unlabeled probe corresponding to the predicted binding sequence of SOT1 on the ndhA transcripts. The asterisk indicates an unspecific band. Three independent experiments were performed, and one representative experiment is shown. ( b ) Overexpressing CAF proteins promoting the binding of SOT1 to the 5′ end of ndhA transcripts in vivo. The HA-tagged CAF1 and FLAG-tagged CAF2 were overexpressed (OE) in protoplasts isolated from 12-day-old seedlings. Chloroplasts were harvested from the transfected protoplasts. Protein complexes were immunoprecipitated (IP) using an α-SOT1 antibody. (Left) Immunoblot analysis of the protein presence in chloroplast extract as immunoprecipitated using an α-SOT1 antibody. The immunoblot results showed a comparable enrichment of SOT1 protein in samples. (Right) The relative levels of psbA , psbB , and ndhA 5′-end mRNAs in the α-SOT1 immunoprecipitated complexes were determined using a qPCR. Student’s t -test was carried out to determine the significance of the fold enrichments of SOT1 on ndhA 5’-end RNAs between WT and OE.CAF protoplast. ** indicates a significant difference at p < 0.01. Mean values ± SD of the triplicate replicates are shown. ( c ) The loss of CAF proteins leads to the decreased association of SOT1 with the 5′ end of ndhA transcripts. The stromal extracts from wild-type, caf1 , caf2 , and caf1 CAF2 -interference (RNAi) plants were immunoprecipitated with an α-SOT1 antibody. (Upper) Immunoblot analysis of the protein presence in the chloroplast extract, as immunoprecipitated using an α-SOT1 antibody. (Lower) The relative levels of psbA , psbB , and ndhA 5′-end mRNAs in the α-SOT1 immunoprecipitated complexes were determined using qPCR. Mean values ± SD of the triplicate replicates are shown.

Article Snippet: The bound protein was eluted with 60 μL of 0.5 mg mL –1 3 × FLAG peptide (MBL, Minato-Ku, Japan) for 1 h. After elution, the proteins were separated using SDS-PAGE and detected using an anti-HA and anti-FLAG immunoblot.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Sequencing, Synthesized, Labeling, Recombinant, Incubation, In Vivo, Isolation, Transfection, Immunoprecipitation, Western Blot