Journal: PLoS Genetics

Article Title: Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis

doi: 10.1371/journal.pgen.1006805

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: p2041 , pRS426 GAL1-TDP43-GFP , Addgene plasmid 27467.

Techniques: Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) Representative images and schematic summary of phenotypes in interphase and mitotic wildtype and TRIM37Δ RPE1 cells. Loss of TRIM37 leads to the formation of a centrobin-containing condensate. Upon mitotic entry, centrobin condensates form ectopic spindle poles at an appreciable frequency, leading to elevated rates of chromosome missegregation. (B) Imaging of a TRIM37Δ RPE1 cell expressing mNG-tagged CEP192 to mark the centrosomes and mRuby-tagged centrobin; SiR-DNA was added to visualize chromosomes—schematics on the right aid in the interpretation of the images. Since TRIM37Δ cells typically have a single condensate, following mitosis, the daughter cell on the left inherits the condensate, while the one on the right (boxed with a dashed line in the 54 min panel ) does not. The cell that did not inherit the condensate was imaged live ( row below ); the region containing the centrosome is magnified on the right; top row is a merge of the CEP192 centrosome marker and centrobin; bottom row shows only centrobin. Centrosomes, visualized by CEP192, are indicated with yellow arrows and the newly forming centrobin condensate with cyan arrowheads. Times are relative to metaphase in the mother cell. A similar phenomenon was observed in 4 cells. (C) ( left ) Schematic summary of the approach used to inducibly knockout TRIM37 (see also ); ( middle ) immunofluorescence images of an inducible TRIM37 knockout ( iTRIM37KO ) cell line without and with doxycycline-induced, Cas9-mediated knockout. Cells were stained for centrobin and the centriolar marker CPAP. Centrosomes are magnified to the left of the lower magnification view and, when present, centrobin condensates to the right. ( right ) Graph plotting the frequency of centrobin condensates for the indicated conditions. n is the number of cells analyzed. (D) ( top left ) Experimental scheme for analyzing the effect of knocking out TRIM37 in cells lacking centrosomes, following PLK4 inhibition with centrinone, and images of cells treated as indicated and labeled for centrobin and CPAP; absence of focal CPAP staining indicates absence of centrioles (see also ). A magnified view of the condensate shows centrobin staining on top ( magenta ) and CPAP signal, which is absent, on the bottom. ( right ) Graph comparing the frequency of condensate formation for the indicated conditions. n is number of cells analyzed. Scale bars are 5µm in panels showing lower magnification views and 1 µm for centrosome and condensate blowups.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Imaging, Expressing, Marker, Knock-Out, Immunofluorescence, Staining, Inhibition, Labeling

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) Schematic of PLK4 highlighting key domains and location of the antigen used to generate ODAb207 in rabbits. Following serum recirculation on the antigen column, antibodies were eluted first using low pH (Acid eluction) and second using high pH (Base elution). The pH of eluted fractions was rapidly neutralized and the Acid and Base elutions were separately dialyzed into a storage buffer. (B) Analysis of ODAb207 staining following inducible knockout of PLK4 in TRIM37Δ p53sh RPE1 cells. Knockout efficiency was confirmed by the absence of centrioles ( not shown ). Two examples are shown for the Acid and Base ODAb207 elutions. For each elution, the top example shows labeling (strong or weak) with ODAb207 of condensates marked by centrobin, while the bottom example shows no labeling. The Base but not the Acid elution of ODAb207 robustly labeled centrobin condensates. Scale bars, 5 µm and 1 µm. (C) Graphical summary of ODAb207 staining of centrobin condensates. Only condensates in acentriolar cells, indicative of PLK4 knockout, were analyzed. Condensate labeling was prominent with the Base elution of ODAb207, even 9 days after knockout induction; by contrast, little-to-no labeling was observed with the Acid elution of ODAb207. n is the number of cells analyzed. The analysis of the inducible PLK4 knockout and the predominant labeling of centrobin-containing condensates with the Base but not Acid elution of ODAb207 indicate that the observed staining does not represent PLK4 and instead represents cross-reactivity with a condensate component. (D) Sequencing traces of control (ODCL192) and iTRIM37 KO (ODCL623) cells following 3 days of doxycycline induction of Cas9. The sequence of the TRIM37 gRNA is indicated above the control sequencing trace. The frequency of indels was estimated using TIDE analysis (see Methods) and is derived by subtracting the percentage of 0 bp changes (10.4%) from 100%. (E) Immunoblot of the cell lines shown in (D) highlighting significant reduction in TRIM37 protein after knockout induction. α-tubulin serves as a loading control. (F) Quantification of centriole depletion following 5-day centrinone treatment of iTRIM37 KO cells. Centrinone was used to first deplete centrioles prior to inducing TRIM37 knockout. n is the number of cells analyzed.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Staining, Knock-Out, Labeling, Sequencing, Control, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) AlphaFold model of the anti-parallel TRIM37 dimer. The RING and B-box-2 domains are on opposite ends of the dimer, and the TRAF domains are positioned just below the mid-point of the anti-parallel coiled-coil. One TRIM37 monomer is colored from the N-terminus in blue to the C-terminus in red; the second TRIM37 monomer is colored grey. See also . (B) ( left ) Schematic of TRIM37 along with an AlphaFold model of the TRAF domain highlighting the tryptophan residue (W373) that was mutated to prevent putative peptide ligand engagement (see also ). ( right ) Immunoblot of TRIM37Δ RPE1 cells engineered to express the indicated TRIM37 variants using lentiviral transduction. The TRIM37 transgenes include a FLAG tag, which is immunoblotted; α-tubulin serves as a loading control. Numbers below the lanes indicate the percentage of cells in the transduced pool that express the transgene, as assessed by anti-FLAG immunostaining. (C) & (D) Images of control and TRIM37Δ RPE1 cells ( C ) and of TRIM37Δ cells expressing the indicated TRIM37 transgenes ( D ). Cells were labeled for centrobin and DNA; centrosomes ( yellow arrows ) and condensates ( cyan arrowhead s) are indicated on the images. Centrosomes are magnified to the left of the lower magnification view and, when present, centrobin condensates to the right. (E) Frequency of centrobin condensate formation for the indicated conditions. n is the number of cells analyzed. (F) ( left ) Experimental schematic of analysis of TRIM37 interaction with centrobin following co-expression in FreeStyle 293F cells. The ligase activity of TRIM37 was mutated to enable robust expression and assessment of binding. The centrobin fragment (1-767) is soluble and contains the TRIM37-binding region (see & ), facilitating the binding analysis. ( right ) Immunoblot of Centrobin (1-767), detected using the Myc epitope tag, and TRIM37, detected using the FLAG epitope tag, in cell lysates and following anti-Myc immunoprecipitation. α-tubulin serves as a loading control for the input lysates. Scale bars in panels C and D , 5 µm ( lower magnification views ) and 1 µm ( centrosome and condensate blowups ).

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Residue, Western Blot, Transduction, FLAG-tag, Control, Immunostaining, Expressing, Labeling, Activity Assay, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) Predicted Aligned Error (PAE) plot of the TRIM37 dimer model. (B) TRIM37 dimer model colored by confidence (pLDDT score; left ) and by chain ( right ). (C) TRIM37 TRAF domain model colored by confidence (pLDDT score).

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques:

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) ( left ) Schematic of full-length (FL) and engineered centrobin fragments; cc refers to predicted coiled-coils. ( right ) Immunoblot analyzing binding of TRIM37 to centrobin fragments, performed as in . Note that the input shown here is the crude extract, prior to centrifugation. For FL centrobin, which is largely insoluble, the clarified supernatant used for the anti-Myc immunoprecipitation is depleted of the TRIM37 ligase-mutant, which pellets with the insoluble centrobin assemblies; this explains the absence of a TRIM37 band in the FL centrobin immunoprecipitation. Similar results were observed in two independent experiments. (B) ( left ) Sequence of the TRIM37-binding region of centrobin, highlighting potential TRAF domain-binding motifs and mutations engineered to disrupt them; ( right ) AlphaFold models of centrobin motifs interfacing with the TRIM37 TRAF domain. The TRAF domain is shown in a space-filling view in gray; the motifs are colored by the AlphaFold prediction confidence score (see also ). Similar results were observed in two independent experiments. (C) Analysis of TRIM37 binding to wildtype or a mutant form of centrobin (1-767) in which the putative TRAF-binding motifs were mutated as indicated in . The binding assay was conducted as in . (D) ( left ) Coomassie-stained gel showing purified recombinant WT versus W373A-mutant TRAF domains ( arrow ). Both lanes shown are from the same gel; an intervening lane was removed ( white line ). ( right ) Analysis of binding of the indicated fluorescent peptides to the purified TRAF domains, monitored using fluorescence polarization (see also ). (E) ( left ) In vivo comparison of wildtype or TRIM37 binding motif-mutant centrobin expressed in either TRIM37Δ;CNTROBΔ or TRIM37 WT; CNTROBΔ cells (see also ). The centrobin transgenes included an mRuby tag for visualization. ( right ) Quantification of the frequency of centrobin condensate formation for the indicated conditions. n is the number of cells analyzed. Scale bars are 5µm in panels showing lower magnification views and 1 µm for centrosome and condensate blowups.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Western Blot, Binding Assay, Centrifugation, Immunoprecipitation, Mutagenesis, Sequencing, Staining, Purification, Recombinant, Fluorescence, In Vivo, Comparison

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) PAE plots of TRIM37 TRAF–centrobin motif models. The regions of TRIM37 and centrobin used to generate the models are noted above. (B) TRAF-centrobin motif models ( same as in ) with binding affinities of FITC-coupled motif peptides estimated from the curves shown in . (C) Sequence trace of a control cell line highlighting the CNTROB gRNA. (D) Sequencing traces of CNTROBΔ cell lines with TRIM37 WT or TRIM37 Δ. For each cell line, the top trace is with a forward sequencing primer and the bottom trace with a reverse sequencing primer (presented as the complementary strand). The changes in each allele are annotated on the traces. (E) Immunoblot verifying loss of centrobin protein in CNTROBΔ cells, with or without TRIM37 present. α-tubulin serves as a loading control.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Binding Assay, Sequencing, Control, Western Blot

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) Images of CNTROBΔ cells, either without or with TRIM37 present, in which either mRuby-tagged WT or Motif 1-mutant centrobin was expressed from a lentivirally delivered transgene. For TRIM37 WT cells expressing the Motif 1-mutant, example images are shown of cells with and without a condensate. Scale bars, 5 µm and 1 µm. (B) Quantification of centrobin condensate frequency for the indicated conditions. (C) ( top ) Protein sequences of miniTRIM37 RBhcc and Bhcc. ( bottom ) Coomassie-stained gels of purified recombinant miniTRIM37 RBhcc and Bhcc proteins (schematized above the gels; the same schematics are shown in ). The RING mutant migrates higher than the wild-type protein, likely due to the negative charges introduced by the mutations made to disrupt the RING-RING interface. The identity of the RING mutant band was confirmed using mass spectrometry. (D) ( top ) miniTRIM37 RBhcc dimer model, also shown in , and ( bottom ) PAE plot of the miniTRIM37 RBhcc dimer model. The color-coded two chains used to construct the model are shown on the left and bottom sides of the PAE plot. (E) SEC-MALS analysis of miniTRIM37 Bhcc WT and mutant purified proteins. Both proteins elute at the same time from the column and have the same native molecular weight, corresponding to the predicted monomer molecular weight.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Mutagenesis, Expressing, Staining, Purification, Recombinant, Mass Spectrometry, Construct, Molecular Weight

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) ( left ) Schematic of full-length centrobin and fragments highlighting the TRIM37 binding region ( green ); ( right ) analysis of solubility of indicated centrobin variants expressed in Freestyle 293F cells. α-tubulin serves as a loading and solubility control. The blot was overexposed to highlight the signals in the supernatant (FL) and pellets (all other variants). (B) Analysis of centrobin fragment ubiquitination by WT TRIM37 following co-expression in FreeStyle 293F cells followed by immunoprecipitation and immunoblotting. Endogenous ubiquitin was detected in the immunoprecipitates using an anti-ubiquitin antibody. (C) Schematic model of substrate oligomerization-directed activation of TRIM37 ligase activity. (D) Schematics of mini-TRIM37 variants engineered to examine the roles of the RING and B-box-2 domains in oligomerization (see also ). (E) SEC-MALS analysis of purified recombinant miniTRIM37s. The numbers above indicate the predicted molecular weights from the primary sequences and the native molecular weights of the entities in the major UV peaks measured by SEC-MALS.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Binding Assay, Solubility, Control, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Western Blot, Activation Assay, Activity Assay, Purification, Recombinant

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) AlphaFold model of the miniTRIM37 RBhcc dimer, highlighting the predicted RING-RING and B-box-2 – B-box-2 interfaces. (B) Detailed view of the 2 predicted interfaces, highlighting key residues. Mutations were engineered in the residues colored in red to disrupt key interface contact. (C) SEC-MALS analysis of the indicated miniTRIM37 RBhcc variants. Numbers next to the predicted molecular weights are the native molecular weights of the entities in the major UV peaks measured by MALS. The SEC-MALS analysis was repeated twice with identical results (see also ). The WT trace is reproduced from for comparison. (D) Immunoblot of transgene-mediated expression of the indicated engineered TRIM37 variants in TRIM37Δ cells. The lanes shown are from the same gel and immunoblot; one intervening lane was removed, as indicated by the white line. α-tubulin serves as a loading control. The left four lanes are reproduced from for comparison. (E) Immunofluorescence images of TRIM37Δ cells expressing the indicated transgene variants after fixing and staining for centrobin and DNA. Centrosomes (yellow arrows) and centrobin condensates (cyan arrowheads) are marked on the images. Scale bar is 5 µm. (F) Quantification of frequency of centrobin condensate formation for the indicated conditions. n is the number of cells analyzed. (G) Analysis of TRIM37 autoregulation following expression in FreeStyle 293F cells. Similar results were observed in two independent experiments. (H) Analysis of centrobin ubiquitination by the indicated TRIM37 variants. HA-ubiquitin was co-transfected along with FL centrobin and indicated TRIM37 variants. The ubiquitination assessed is on the small pool of soluble FL centrobin following immunoprecipitation. Similar results were observed in two independent experiments.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Comparison, Western Blot, Expressing, Control, Immunofluorescence, Staining, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) ( left ) Schematics highlighting TRIM37 variants expressed from transgenes in TRIM37Δ cells; ( right ) images of cells expressing the TRIM37 variants that were fixed and labeled for centrobin and for the FLAG epitope fused to the transgene-encoded TRIM37 variants. Condensates, when present, are highlighted by cyan arrowheads and are shown in a magnified view on the right, with separated centrobin ( magenta ) and FLAG ( green ) channels. (B) Quantification of the ratio of FLAG to centrobin signal at condensates for the indicated TRIM37 variants. The plotted values were normalized relative to the median value of the TRIM37 ligase-mutant. p-values are from unpaired t-tests; ****: p<0.001. (C) Immunoblot of transgene-mediated expression of the indicated engineered TRIM37 variants in TRIM37Δ cells. α-tubulin serves as a loading control. (D) Model for the substrate-dependent oligomerization and activation of TRIM37. Model highlights how TRIM37 selectively targets oligomers (i) and summarizes the effect of TRIM37 mutations (ii).

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Expressing, Labeling, FLAG-tag, Mutagenesis, Western Blot, Control, Activation Assay

Journal: bioRxiv

Article Title: TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

doi: 10.1101/2024.10.09.617493

Figure Lengend Snippet: (A) ( left ) Schematics of transgene-encoded TRIM37::3xFLAG variants and ( right ) images of TRIM37Δ cells expressing indicated variants and labeled for centrobin and the FLAG epitope. Scale bars, 5 µm & 1 µm. (B) Quantification of the ratio of FLAG to centrobin signal at condensates for the indicated TRIM37 variants. The plotted values were normalized relative to the median value of the TRIM37 ligase-mutant. The data shown for the ligase-mutant is the same as in . p-values are from unpaired t-tests; ****: p<0.0001.

Article Snippet: The TRIM37 TRAF domain coding sequence (residues 274-407) was amplified and cloned into the UC Berkeley Macrolab vector 2CT (Addgene number: 29706) to express N-terminal TEV protease-cleavable His 6 -MBP-tagged fusions.

Techniques: Expressing, Labeling, FLAG-tag, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: Co-expression of snc2 R52Q and a chimeric SPO20 rescues the sporulation defect of sso1 Q224R . Strain HJ3 ( sso1Δ / sso1Δ spo20Δ / spo20Δ ) was transformed with plasmids carrying the indicated genes and sporulated in liquid culture. SSO1 alleles were expressed from CEN plasmids; SNC2 and SPO20 alleles were expressed from high copy plasmids. Sporulation was assessed by observation in the light microscope or by ether test. To determine percentage of sporulation, at least 500 cells were counted for each strain; percentages represent the average of four experiments. “a”, “b”, and “c” illustrate the arrangement of side chain residues at the ionic layer in the different strains.

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: Expressing, Transformation Assay, Light Microscopy

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: Mutation of two interface residues in the Spo20 SNARE helix allows it to function with sso1 Q224R . (A) Alignment of the interface residues in the SNARE domains of Spo20 and Sec9. Residues chosen for mutation are in blocks. (B) Sporulation of sso1 Q224R snc2 R52Q strains expressing different forms of SPO20 . Strain HJ3 ( sso1Δ / sso1Δ spo20Δ / spo20Δ ) was transformed with plasmids carrying sso1 Q224R and snc2 R52Q as well as the indicated form of SPO20 . The sso1 Q224R allele was expressed from a CEN plasmid; snc2 R52Q and the SPO20 alleles were expressed from high copy plasmids. These strains were sporulated and sporulation efficiency measured in the light microscope. At least 500 cells were scored for each strain. Results are the average of three experiments. Error bars indicate one standard deviation.

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation, Light Microscopy, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: SPO20 C224L,S231N can rescue the growth defect of sec9-4 . (A) Strain AN211 ( sec9-4 ) was transformed with integrating or centromeric (for PSPP and PPPS ) plasmids expressing the indicated genes. Cells were grown to saturation in rich medium and 10-fold serial dilutions were spotted onto selective plates incubated at permissive (25°C) or restrictive (37°C) temperature. SPO20 CS,LN is SPO20 C224L , S231N ; SPO20 CSFF,LNTL is SPO20 C224L,S231N,F357T,F361L ; SPO20 CSFFAK,LNTLLN is SPO20 C224L,S231N,F357T,F361L,A378K,K385N . (B) A similar growth assay in strain AN211 ( sec9-4 ) using high copy plasmids to express the indicated genes.

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: Transformation Assay, Expressing, Incubation, Growth Assay

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: Mutation of the Spo20 helices increases binding of Sso1 and Snc2. Strain HI75 ( sso1Δ sso2Δ ) was transformed with high copy plasmids expressing 3xHA tagged forms of Δ3-51Spo20, Δ3-51Spo20 C224L , S231N , or Δ3-51PSPS chimera. Additionally, these cells carried a CEN plasmid expressing either SSO1 or SSO1 Q224R and high copy plasmids expressing SNC2 or SNC2 R52Q , respectively. These strains were grown to mid-log in selective medium, lysed, and the HA-tagged Spo20 proteins immunoprecipitated. (A) (top) Western blots of cell lysates from each strain probed with anti-HA, anti-Sso1, or anti-Snc2 antibodies. 3X-HA indicates bands corresponding to the different Spo20 mutants; (bottom) Western blots of anti-HA immunoprecipitates from the same lysates. The band corresponding to Δ3-51PSPS in the Sso1 Q224R /Snc R52Q strain is shown from a longer exposure of the same blot. Asterisk indicates Sso1 Q224R , which displays slightly increased mobility compared with the wild-type Sso1. (B) Quantitation of the coprecipitation of Sso1 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Sso1 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Sso1 blots. Values shown are the average of three experiments. Bars indicate one standard deviation. (C) Quantitation of the coprecipitation of Snc2 proteins with the different forms of Spo20. Amounts are expressed as the ratio of Snc2 to Spo20 protein based on relative intensity of bands on the anti-HA and anti-Snc2 blots. Values shown are the average of three experiments. Bars indicate one standard deviation.

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: Mutagenesis, Binding Assay, Transformation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Quantitation Assay, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: Spo20 C224L,S231N is a more efficient fusogen in vitro. (A) Liposome fusion assay. Liposomes containing Sso1 and the SNARE domains of Sec9, Spo20, or Spo20 C224L,S231N were mixed with Snc2-containing liposomes and fusion assayed by fluorescence. (B) Stability of Sso1/Snc2/ Spo20 C224L,S231N complexes in increasing concentrations of Guanidinium-HCl.

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: In Vitro, Single Vesicle Fusion Assay, Liposomes, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Binding interactions control SNARE specificity in vivo

doi: 10.1083/jcb.200809178

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: The plasmids pRS426-SPO20pr- SPO20 C224L;S231N , pRS426-SPO20pr- SPO20 A378L;K385N , and pRS426-SPO20pr- SPO20 F357T;F361L were created by site-directed mutagenesis of the plasmid pRS426-SPO20pr- SPO20 ( ) using oligos HJO31 and HJO32, HJO33 and HJO34, and HJO35 and HJO36, respectively.

Techniques: