Journal:

Article Title: Extensive phosphorylation of Smoothened in Hedgehog pathway activation

doi: 10.1073/pnas.0408093101

Figure Lengend Snippet: Smo phosphorylation site variants show transdominant effects in vitro and in vivo. (A) Overexpressed untagged and GFP-tagged Smo variants cause transdominant effects on reporter activity. Either pAcSV- or pUAST-based plasmids expressing the Smo variants shown were transfected in the presence of endogenous Smo. The results of a representative ptc-Luc reporter assay are shown. (B) Overexpression of GFP-tagged Smo-Glu in embryos leads to expansion of Wg expression. Immunofluorescence using anti-GFP (green) and anti-Wg (red) Abs is shown for dorsal views at ×10 and ×25 of embryos at extended germ-band stage that are either wild-type (WT; w1118) or expressing UAS GFP-tagged wild-type (Smo), III,V,VI Ala (Ala), or III,V,VI Glu (Glu) forms of Smo using the prd-GAL4 driver. P ↔ A, orientation of the posterior/anterior axis. (C) Overexpression of Smo variants leads to wing patterning alterations. Wings collected from adult flies that are either wild-type (WT) or heterozygous for the ptc-GAL4 (Upper) or 71B GAL4 (Lower) driver and expressing GFP-Smo variants as in B are shown. Longitudinal veins 1–5 are labeled. Asterisk, proximal L3 and L4 fusion for the GFP-Smo-Ala variant overexpressed with ptc-GAL4; arrowheads, ectopic veination near the proximal end of L3 when GFP-Smo is overexpressed (black) and proximal L3 and L4 fusion when GFP-Smo-Ala is overexpressed (white) using 71B GAL4. Flies expressing GFP-Smo-Glu die as pupae with ptc-GAL4 and as pharate adults with 71B GAL4. A severely defective wing from an escaper from the GFP-Smo-Glu 71B GAL4 line is shown (Lower Right).

Article Snippet: GAL4 driver lines used were prd-GAL4, ptc-GAL4 , and 71B ( 51 ). pUAST-derived plasmids (with the mini white gene) containing GFP-smo coding sequences were injected into w 1118 fly embryos by Genetic Services, Inc. (Sudbury, MA).

Techniques: In Vitro, In Vivo, Activity Assay, Expressing, Transfection, Reporter Assay, Over Expression, Immunofluorescence, Labeling, Variant Assay

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) Schematic of the spatial localization of proteins used as positive mitochondrial control proteins (Atp4, Pam16), negative cytosolic control protein (Pgk1) and as dual localized protein (cERS) in S. cerevisiae . ( B ) Empty pAG414pGPD β11ch vector (EV) or pAG414pGPD β11ch vectors expressing each of the four GFP β11ch -tagged proteins used as markers in our study were transformed into the BiG Mito-Split-GFP strain. cERS β11ch was either expressed under the dependence of the GPD (pGPD) or its own promoter (pGUS1) from a centromeric plasmid. GFP reconstitution upon mitochondrial import was followed by epifluorescence microscopy (N = 3). ( C ) Immunodetection of the GFP β1-10 , cERS β11ch and Pgk1 β11ch fusion protein in whole cell extract from the transformed BiG Mito-Split-GFP strain using anti-GFP and -Pgk1 antibodies, confirming expression of Pgk1 β11ch . Loading control: stain-free. The representative gels are shown. ( D ) The strains described in the legend of panel ( B ) were used for three-dimensional reconstitution of yeast mitochondrial network (N = 1). Z-Stack images from Pam16 β11ch , Atp4 β11ch , cERS β11ch and Pgk1 β11ch were taken using an Airyscan microscope. Scale bar: 1 µm. ( E ) Flow cytometry measurements of total GFP fluorescence of the BiG Mito-Split-GFP strain stably expressing Pgk1 β11ch or Pam16 β11ch (N = 3). ( F ) The mitochondrial GatF protein was fused to the GFP β1-10 fragment (mtGatF β1-10 ), thereby targeting the ten first GFP beta-strands to mitochondria after being transcribed in the nucleus and translated in the cytoplasm. This construct was co-expressed with either cERS β11ch or Pgk1 β11ch . The GFP reconstitution was monitored by epifluorescence microscopy. Mitochondria were stained with MitoTracker Red CMXRos. Scale bar: 5 µm. Representative fields are shown. Figure 2—source data 1. Micrographs of the BiG Mito-Split-GFP expressing Pgk1 β11ch , cERS β11ch , Pam16 β11ch , (related to ). The micrograph of the BiG Mito-Split-GFP expressing Pgk1 β11ch which is magnified in is presented here with adjusted or enhanced contrast settings. A new panel of the BiG Mito-Split-GFP expressing Pgk1 β11ch was added with enhanced or adjusted contrast settings. Figure 2—source data 2. Confirmation of the expression of the GFP β1-10 , cERS β11ch and Pgk1 β11ch fusion proteins in whole cell extract from the transformed BiG Mito-Split-GFP strains (Related to ). Antibodies used for immunoblotting are indicated below WBs. Loading control corresponds to the gel stained with the stain-free procedure. Figure 2—source data 3. Flow cytometry measurements of total GFP fluorescence of the three biological replicates of the BiG Mito-Split-GFP strain stably expressing Pgk1 β11ch or Pam16 β11ch (related to ).

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Control, Plasmid Preparation, Expressing, Transformation Assay, Epifluorescence Microscopy, Immunodetection, Staining, Microscopy, Flow Cytometry, Fluorescence, Stable Transfection, Construct, Western Blot

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) The amino acid sequence and numbering of the residues of wild type GFP β1-10 are shown. The β-strands are schematized as blue arrows. The amino acid residues of wild type GFP that were mutated to generate the Folding Reporter GFP are in green. The six amino acids of Folding reporter GFP that were then mutated to build the Superfolder GFP are in red and the seven amino acid residues of Superfolder GFP that were mutated to generate GFP β1-10 OPT are indicated in orange . ( B ) The amino acid sequence of the GFP β11ch is shown and the numbering corresponds to the aa residues of the β11-strand of wild type GFP. The three consecutive β11 strands are schematized as green arrows and the three mutations that were introduced into each β11 strand (GFP11M3) are in purple . The linker sequences are colored gray.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Sequencing

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) Principle of the Split-GFP system. When present in the same subcellular compartment, two fragments of GFP namely GFP β1-10 and GFP β11ch can auto-assemble to form a fluorescent BiG Mito-Split-GFP chaplet (three reconstituted GFPs). GFP β1-10 sequence encoding the first ten beta strands of GFP has been integrated into the mitochondrial genome under the control of the ATP6 promoter. GFP β11ch consists of a tandemly fused form of the eleventh beta strand of GFP and is expressed from a plasmid under the control of a strong GPD promoter (pGPD). The molecular weight of the tag is indicated. ( B ) Growth assay on permissive SC Glu plates, respiratory plates (SC Gly), and restrictive media lacking arginine (SC Glu -Arg) of the different strains used in the study (N = 2). All generated strains are derivative from MR6. ( C ) ATP synthesis rates of the MR6 and RKY112 strains presented as the percent of the wild type control strain (N = 2). P-value was 0.7456 (not significant). 95% confidence interval was −273.4 to 229.9, R squared = 0.064 ( D ) Mitochondrial translation products in the MR6 and RKY112 strains (N = 2). Cells were grown in rich galactose medium. Pulse-chase of radiolabeled [ 35 S]methionine + [ 35 S]cysteine was performed by a 20 min incubation in the presence of cycloheximide. Total cellular extracts were separated by SDS PAGE in two different polyacrylamide gels prepared with a 30:0.8 ratio of acrylamide and bis-acrylamide. Upper gel: 12% polyacrylamide gel containing 4 M urea and 25% glycerol. Lower gel: 17.5% polyacrylamide gel. Gels were dried and exposed to X-ray film. The representative gels are shown. Figure 1—source data 1. Respiratory competency and translation of mtDNA-encoded respiratory subunits of the strains used in this study. Growth assay on permissive SC Glu plates, respiratory plates (SC Gly), and restrictive media lacking arginine (SC Glu -Arg) of the different strains used in the study (related to ). Mitochondrial translation products in the MR6 and RKY112 strains (N = 2) monitored by pulse-chase labeling with radiolabeled [ 35 S]methionine and [ 35 S]cysteine (related to ). Figure 1—source data 2. Statistics of the comparison of ATP synthesis rates between RKY112 and MR6 strains (related to ).

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Sequencing, Control, Plasmid Preparation, Molecular Weight, Growth Assay, Generated, Pulse Chase, Incubation, SDS Page, Labeling, Comparison

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: Genotypes of yeast strains used or generated for this study.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Generated

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) Colocalization measurement of the reconstituted GFP ( β11+ β1-10 ) with MitoTracker Red CMXRos-stained mitochondria on merged micrographs shown in . Fluorescent signals were measured along the yellow line with the ImageJ software. ( B ) Fluorescence microscopy analysis of the BiG Mito-Split-GFP strains bearing integrated into the TRP1 locus of GUS1 (cERS), PAM16 or PGK1 genes fused to GFP β11ch . The cERS β11ch is expressed from the own promoter ( GUS1 ) while Pam16 β11ch and Pgk1 β11ch are expressed from GPD promoter. The last panel (Pgk1 β11ch Increased brightness) shows the full field from which the Pgk1 β11ch micrograph of the upper panel was taken from, with enhanced brightness, thereby illustrating the absence of any faint mitochondrial fluorescence. Mitochondria were stained with MitoTracker Red CMXRos. Scale bar: 5 µm.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Staining, Software, Fluorescence, Microscopy

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: Fluorescence microscopy analyses of BiG Mito-Split-GFP strain transformed with pAG414pGPD β11ch expressing yeast caaRSs (also see Table S3). Genes encoding 18 out of the 20 yeast caaRS, including those encoding the α- and β-subunits of the cytosolic α 2 β 2 FRS (cFRS2), and the cGRS2 pseudogene, as well as the four encoding the cytosolic echoforms of cGRS1 ( cyte cGRS1), cARS ( cyte cARS), cHRS ( cyte cHRS) and cVRS ( cyte cVRS) were cloned in the pAG414pGPD β11ch and expressed in the BiG Mito-Split-GFP strain (N = 2). ( A ) From the set of caaRSs tested, only cERS, cQRS, cFRS2 and cyte cHRS micrographs are shown. ( B ) Table summarizing the GFP emission and mitochondrial localization of the caaRSs not shown in A ). The corresponding micrographs are shown in Fig. S4A. ( C ) Fluorescence microscopy analysis of the BiG Mito-Split-GFP strain expressing the first 100 amino acids of the N-ter region of the cCRS fused to GFP β11ch (N = 2). ( D ) Fluorescence microscopy analyses of BiG Mito-Split-GFP strain transformed with pAG414pGPD β11ch expressing the mitochondrial echoforms mte cGRS1, mte cARS, mte cHRS and mte cVRS. Schematics of cARS, cGRS1, cHRS and cVRS echoforms expression in yeast. Expression can be initiated upstream of the initiator ATG +1 ( mte cARS at ACG -75 and mte cGRS1 at TTG -69 ) but the synthesis of this echoform can also be initiated at the ATG +1 . In this case, the expression of the cytosolic echoform is initiated downstream ( cyte cHTS at ATG +60 and cyte cVRS at ATG +148 ). Mitochondria were stained with MitoTracker Red CMXRos. Scale bar: 5 µm. Representative fields are shown. Figure 3—source data 1. Confirmation, by WB, of the expression of the 18 full-length aaRS β11ch and N100cCRS β11ch in whole cell extracts from the transformed BiG Mito-Split-GFP strains (Related to ). Antibodies used for immunoblotting are indicated below WBs. Loading controls correspond to gels stained with the stain-free procedure.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Fluorescence, Microscopy, Transformation Assay, Expressing, Clone Assay, Staining, Western Blot

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) Micrographs of all the other caaRSs tested in . Representative panels from two independent experiments are shown. Mitochondria were stained with MitoTracker Red CMXRos. Scale bar: 5 µm. Representative fields are shown. ( B ) Immunodetection of all the GFP β11ch -tagged aaRSs expressed in the BiG Mito-Split-GFP strain. aaRS β11ch were detected by anti-GFP antibodies. Equal loading was verified by anti-Pgk1 antibodies and by stain-free technology (Loading control). caaRS: cytosolic aaRS, cyte caaRS: cytosolic echoform of the caaRS, mte caaRS: mitochondrial echoform of the caaRS.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Staining, Immunodetection, Control

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A ) Schematic representation of the cERS fragments fused to GFP β11ch . Orange boxes correspond to the GST-like domain necessary for Arc1 interaction (GST), the grey boxes represent the catalytic domain (CD), and the blue box, the tRNA-binding domain generally named anti-codon binding domain (ABD). Numbering above corresponds to cERS amino acids residues. ( B ) Fluorescence microscopy analyses of the BiG Mito-Split-GFP strain expressing the cERS variants shown on A . Mitochondria were stained with MitoTracker Red CMXRos; scale bar: 5 µm. The secondary structure (according to ) of the smallest peptide that still contains the non-conventional MTS of cERS is described together with the amino acid sequence of each helices. Positively and negatively charged amino acids are shown in orange and blue respectively. ( C ) Immunodetection of the cERS variants in BiG Mito-Split-GFP whole cell extracts using anti-GFP antibodies. Quantity of proteins loaded in each lane was estimated using anti-Pgk1 antibodies or by the stain-free procedure. The bands corresponding to the mutants N1 and N2 could not be detected. The representative fields or gel are shown. Figure 4—source data 1. Immunodetection of the cERS variants in BiG Mito-Split-GFP whole cell extracts using anti-GFP antibodies (related to ). Antibodies used for immunoblotting are indicated below WBs. Loading controls correspond to gels stained with the stain-free procedure.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Binding Assay, Fluorescence, Microscopy, Expressing, Staining, Sequencing, Immunodetection, Western Blot

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet: ( A, D ) Prediction of MTS and mitochondrial localization of ( A ) two ERS from Arabidopsis thaliana ( Ath cERS and Ath mt/chlERS) and ( D ) five eukaryotic Ago2 proteins [ Hsa Ago2 (Protein argonaute-2 isoform X2 [Homo sapiens] NCBI sequence ID: XP_011515267.1), Mmu Ago2 (protein argonaute-2 Mus musculus NCBI sequence ID: NP_694818.3.), Bta Ago2 ( Bos Taurus ), D re Ago2 ( Danio rerio ), Dme Ago2 ( Drosophila melanogaster ). MTS were predicted using TPpred2.0 ( http://tppred2.biocomp.unibo.it/tppred2 ), TargetP1.1 ( http://cbs.dtu.dk/services/TargetP/ ), MitoFates ( http://mitf.cbrc.jp/MitoFates/cgibin/top.cgi ) and the EukmPloc2 website ( http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/ ). Grey boxes indicate prediction of a cytosolic localization, light and dark green indicate prediction of mitochondrial or chloroplastic localization respectively. Blue boxes indicate prediction of nuclear localization. ( B, E ) Fluorescence microscopy analyses of the BiG Mito-Split-GFP strain expressing the GFP β11ch -tagged Ath cERS and Ath mt/chlERS (N = 2) ( B ) and Mmu Ago2, Hsa Ago2 (N = 2) ( E ). Mitochondria were stained with MitoTracker Red CMXRos. Scale bar: 5 µm. Representative fields are shown. ( C, E ) Protein expression was checked by WB with anti-GFP antibodies and equal amount of loaded protein was controlled using anti-Pgk1 antibodies and by the stain-free technology (Loading control: stain-free). The representative gels are shown. Figure 5—source data 1. Confirmation, by WB, of the expression of AthERS β11ch and mouse and human Ago2 β11ch in whole cell extract from the transformed BiG Mito-Split-GFP strains (Related to ). Antibodies used for immunoblotting are indicated below WBs. Loading controls correspond to gels stained with the stain-free procedure.

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Sequencing, Fluorescence, Microscopy, Expressing, Staining, Control, Transformation Assay, Western Blot

Journal: eLife

Article Title: Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

doi: 10.7554/eLife.56649

Figure Lengend Snippet:

Article Snippet: The GFP β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB β11ch .

Techniques: Clone Assay, Recombinant, Plasmid Preparation, Construct, Cloning, Staining, Control