Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) Illustration of the spindle position checkpoint in budding yeast and localization of the MEN GTPase Tem1 and its GAP. (B) Major components of the MEN and its inputs and outputs. (C) Molecular switch model for Tem1. (D) GTPase activity of Tem1. Recombinantly purified Tem1 was mixed with GTP and [α- 32 P]GTP on ice and the reaction was initiated by shifting to 30 °C. Samples were taken at indicated time points and analyzed by TLC . Shown are the average of 3 technical replicates with a linear fit. Error bars represent ± SD. (E) The rate of nucleotide exchange (MANT-GDP to GTP) for Tem1 and N-Ras. Recombinantly purified Tem1 (5 µM final) and N-Ras was first incubated with MANT-GDP (200 nM final) and the nucleotide exchange was initiated by the addition of excess GTP (1 mM). Shown are the average of 3 technical replicates with an exponential fit for Tem1 and a linear fit for N-Ras. Error bars represent ± SD. (F) The binding affinity of Tem1 for MANT-GDP. 200 nM MANT-GDP was titrated with increasing amount of purified apo-Tem1. Shown are the average from 3 technical replicates with a quadratic fit for the dissociation constant ( K D ). Error bars represent ± SD.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Activity Assay, Purification, Incubation, Binding Assay

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) Purification of Tem1. Tem1 was expressed in E. coli as a 14xHis-SUMO-Tem1 fusion with p2579. Lane 1 , lysate; lane 2 , soluble fraction; lane 3 , insoluble fraction; lane 4 , flowthrough of Ni-NTA column; lane 5 , wash with lysis buffer; lane 6 , elution after overnight digest with SENP protease which contains cleaved Tem1; lane 7 , elution with high imidazole buffer (250 mM imidazole) where the remaining 14xHis-SUMO and uncut fusion protein were eluted; lane 8 , input for Mono S column; lane 9 , wash with buffer A; lane 10 , combined peak elution of Tem1 from Mono S. Arrowhead marks 14xHis-SUMO-Tem1 fusion (41 kDa) and arrow marks Tem1 (27 kDa). (B) An example of raw thin layer chromatography (TLC) results for the GTPase activity of recombinantly purified Tem1 and the hydrolysis deficient mutant Tem1Q79L. No protein was added to the control sample (buffer only). (C) An example of raw data for . (D) Summary of biochemical properties of Tem1 in vitro . Shown are the averages of three independent experiments and SD in parentheses. n.d., not determined (due to GTP hydrolysis). The difference of the observed exchange rate between MANT-GTP and MANT-GTPγS ( k obs ≈ k off (GTP or GTPγS) + k hydrolysis ) is likely not driven by GTP hydrolysis as previously assumed in filter-binding assays because GTP hydrolysis occurs at a rate an order of magnitude slower than dissociation ( k obs ∼ 10 - s - , while k hydrolysis ∼ 10 - s - ). Rather, we think that the observed rates reflect differences in affinity or off rate of the nucleotides ( k obs γ k off , when k hydrolysis << k obs ). The higher affinity for MANT-GTPγS relative to MANT-GDP is consistent with the lower exchange rate for MANT-GTPγS we observed.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Purification, Lysis, Thin Layer Chromatography, Activity Assay, Mutagenesis, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: ( A-C ) Representative images and quantification of SPB localization in wild-type (y1748, n = 73 cells), GTP-locked hydrolysis mutant Q79L (y1824, n = 63 cells), GDP-locked/apo nucleotide binding mutant T34A/N (y3167/y3171, n = 29/21 cells), and effector binding mutant T52A (y3147, n = 32 cells) of Tem1. Cells expressing tem1T34A/N and tem1T52A (marked with *) were kept alive with the hyperactive DBF2-HyA that can rescue tem1Δ . Cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Single cell traces were aligned based on anaphase onset, as defined as spindle length > 3 μm (measured based on SPB marker Spc42-mScarlet-I), and averaged. Solid lines represent the mean, shaded areas represent 95% confidence intervals. For maximum enrichment at SPB, solid lines represent the median. **** p < 0.0001 by two-sided Wilcoxon rank sum test for comparing with WT . (D) Complementation analysis of Tem1 mutants with and without tethering to the SPB via CNM67-GBP . 5-fold serial dilutions of strains y2891/y3202/y3203/y3204/y3214 and y3303/y3304/y3305/y3306/y3310, with the indicated TEM1 alleles, were spotted onto plates with or without 5’-fluoroorotic acid (5-FOA) and incubated at 25°C for 2 to 3 days. The presence of 5-FOA selects cells that are viable after losing the TEM1 (URA3/CEN) plasmid. GBP = GFP binding protein. (E) Distribution of anaphase duration for different yEGFP - TEM1 alleles in the presence of CNM67-GBP (y3289, y3287, and y3291; n =46, 43, and 47 cells respectively) measured using the SPB marker Spc42-mScarlet-I. Cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the median.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Mutagenesis, Binding Assay, Expressing, Marker, Incubation, Plasmid Preparation

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) Mutated residues in Tem1. A model of Tem1 (residues 13–187 containing the core GTPase domain) was downloaded from Swiss Model (P38987_20210204) using Rab35 (GTP)-RUSC2 as a template. Tem1 backbone (gray ribbon), GTP (cyan), Mg 2+ (dark gray sphere), P-loop with T34 (yellow), switch-I with T52 (purple), switch-II with G78 (orange) and D75 (green). T34, T52, and D75 coordinate the Mg 2+ atom while G78 forms hydrogen bond with the γ-phosphate (dashed lines). T34 (Ras-S17) and D75 (Ras-D57) contribute to nucleotide binding and mutations in these residues have been shown to bias affinity toward GDP over GTP for Ras. T52 (Ras-T35) and G78 (Ras-G60) are invariant residues in switch I/II regions of small GTPases that form key contacts with the γ-phosphate in GTP to stabilize the active confirmation that is used by regulatory proteins and effectors to ‘sense’ the nucleotide status of the small GTPases. (B) Complementation analysis of Tem1 mutants with and without tethering to the SPB via direct fusion to Cnm67 (Cnm67-Tem1) or using yGFP-Tem1 + Cnm67-GBP combo. 5-fold serial dilutions of strains y2891/y3202/y3203/y3204/y3206/y3207/y3214/y3205 (untethered) and y2891/y2893/y2927/y3187/y3190/y3191/y3189/y3188 (direct fusion as Cnm67-Tem1) or y3303/y3304/y3305/y3306/y3308/y3309/y3310/y3307 (with CNM67-GBP ) were spotted onto plates with or without 5’-fluoroorotic acid (5-FOA) and incubated at 25°C for 3 to 4 days. The presence of 5-FOA selects cells that are viable after losing the TEM1 (URA3/CEN) plasmid. Mutations in T34, D75, and T52 render Tem1 inactive and severely reduce viability. In contrast, the switch-II effector mutant Tem1G78A is active.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Binding Assay, Incubation, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) SPB localization of Tem1 or the GTP-locked Tem1Q79L with or without the GAP component BFA1 and the anaphase duration for the corresponding cells. Cells of y2015 (Tem1, n = 54 cells), y2098 (Tem1 in bfa1≈ , n = 64 cells), y2019 (Tem1Q79L, n = 79 cells), and y2133 (Tem1Q79L in bfa1≈ , n = 64 cells) were grown at 25 °C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the median. ns , not significant ( p > 0.05), * p < 0.05 by two-sided Wilcoxon rank sum test. (B) SPB localization of Tem1 or the GTP-locked Tem1Q79L with or without the GAP component BUB2 and the anaphase duration for the corresponding cells. Cells of y1748 (Tem1, n = 65 cells), y1929 (Tem1 in bub2≈ , n = 72 cells), y1824 (Tem1Q79L, n = 91 cells), and y1930 (Tem1Q79L in bub2≈ , n = 64 cells) were grown at 25 °C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. For SPB localization, solid lines represent the average. Shaded areas represent 95% confidence intervals. For anaphase duration, solid lines represent the median. ns , not significant ( p > 0.05), ** p < 0.01 by two-sided Wilcoxon rank sum test. (C) SPB localization of Tem1 with or without BFA1 & BUB2 . Cells of y2015 (Tem1, n = 44 cells), y3717 (Tem1 in bfa1≈+bub2≈ , n = 42 cells), and y3718 (Tem1 in bub2≈ , n = 50 cells) were grown at 25 °C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the average. Shaded areas represent 95% confidence intervals. (D) Comparison of SPB localization for Tem1 and Cdc15 in the presence or absence of BUB2 . Same data as in .

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Comparison

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: ( A-B ) Interaction between different Tem1 mutants and the GAP complex (A) or Cdc15 (B) via co-immunoprecipitation. yEGFP-Tem1 was immunoprecipitated from exponentially grown cells of the indicated genotypes (y3461/y3452/y3472/y3457/y3456 or y2761/y2885/y2886/y3209/y3208) and the presence of Bfa1-3V5 or Cdc15-3HA were analyzed by Western blot analysis. ( C ) Illustration of the hybrid AlphaLISA assay used in this study. Recombinant biotinylated Tem1 were incubated together with cell lysate with eGFP tagged binding partners in the presence of GTPγS or GDP. The interaction was monitored with streptavidin coated Alpha donor beads and anti-GFP conjugated accepter beads. ( D-E ) Interaction between recombinant biotinylated Tem1 and eGFP tagged Bub2 (D) or Cdc15 (E) in lysate monitored by AlphaLISA. Diluted lysate of y1378 (Bub2-eGFP) or y1275 (Cdc15-eGFP) were incubated with different concentrations of recombinant biotinylated Tem1 in the presence of GTPγS or GDP. The Alpha signals were normalized to the maximum signal of each sample in the GTPγS condition. Black lines represent the average of three biological replicates and error bars denote standard deviation (SD). The Hook point is when the biotin-Tem1 added exceeds the binding capacity of the Alpha Donor beads which results in a competition between biotin-Tem1 in solution and on beads for binding. Red line indicates 10% of the available eGFP tagged protein (maximum binding capacity defined by the GTPγS condition) were bound by biotin-Tem1 on beads. (F) Dissociation constant of Tem1 binding to Cdc15 and the GAP estimated by a competition binding assay. Diluted lysate of y1378 (Bub2-eGFP) or y1275 (Cdc15-eGFP) were incubated with a fixed amount of biotinylated Tem1 with different concentrations of unlabeled Tem1 in the presence of GTPγS or GDP. Signal for Bub2-eGFP with GDP was too low to be quantified reliably. The Alpha signals were double normalized to the signal without competing Tem1 (normalized signal = 1) and without biotin-Tem1 (normalized signal = 0). Lines represent the average of three biological replicates and error bars denote SD. ( G-H ) Interaction between biotinylated Tem1 and eGFP tagged Bub2 (G) or Bfa1 (H). Diluted lysate of y1374 (Bfa1-eGFP), y1378 (Bub2-eGFP), y3099 (Bfa1-eGFP in bub21′ ), or y3632 (Bub2-eGFP in bfa11′ ) were incubated with different concentrations of biotinylated Tem1 in the presence of GTPγS or GDP, and the interaction was monitored with AlphaLISA. Lines represent the average of three biological replicates and error bars denote SD.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Immunoprecipitation, Western Blot, Recombinant, Incubation, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) Illustration of the different routes for Tem1 and the GTP-locked Tem1Q79L at dSPB. Once Tem1 GTP is recruited to the dSPB by the GAP, its departure can occur either via Tem1 GTP dissociation ( k off T ) or GAP-stimulated GTP hydrolysis ( k hyd ) followed by dissociation ( k off D ). Assuming k off D >> k hyd , the residence time of Tem1 at the dSPB ( t 1/2 Tem ) is set by the sum of two rates: t 1/2 Tem = ln2/( k off T + k hyd ). For Tem1Q79L, its residence time ( t 1/2 Tem1Q79L ) is simply t 1/2 Tem1Q79L = ln2/ k off T assuming that Tem1 GTP and Tem1Q79L dissociate at similar rates. (B) FRAP analysis of Tem1 and TemQ79L at dSPB in anaphase. Cells of y3900 ( TEM1 , n = 9 cells) and y3903 ( TEM1Q79L , n = 15 cells) were grown and imaged at room temperature in SC medium + 2% glucose + 100 μM IAA. Circles represent the average normalized fluorescence intensities after correcting for photo-bleaching during acquisition. Solid lines are the average fit and shaded areas represent SD. Half recovery time t 1/2 ± SD are indicated. (C) FRAP analysis of Tem1 at dSPB in metaphase and anaphase. Cells of y3900 were grown and imaged at room temperature in SC medium + 2% glucose + 100 μM IAA. (D) dSPB localization of Tem1 in cells with SPB-tethered GAP. Cells of y2015 ( BFA1 , n = 54 cells), y2098 ( bfa1Δ , n = 64 cells), and y2990 ( SPC72-BFA1 , n = 48 cells) were grown at 25 °C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the average. Shaded areas represent 95% confidence intervals. (E) Proposed model for GAP and Tem1 localization at the SPB. Under wildtype condition (low [Tem1 GTP ] in the cell), only a fraction of the GAP complex at the dSPB is bound to Tem1 GTP in both metaphase and anaphase. While more GAP complex is recruited to dSPB in anaphase, the fraction of GAP bound to Tem1 GTP does not change between metaphase and anaphase. In cells with high [Tem1 GTP ] such as with TEM1Q79L , most of the GAP complex are bound to [Tem1 GTP ]. (F) dSPB localization of Tem1 ( WT or Q79L ) and Bfa1 (expressing TEM1 or TEM1Q79L ) in cells with Cdc15 depletion. Cells of y3900 ( TEM1 , n = 16 cells), y3903 ( TEM1Q79L , n = 35 cells), y4123 (Bfa1 in TEM1 , n = 24 cells), and y4122 (Bfa1 in TEM1QL , n = 32 cells) were grown at 25 °C in SC medium + 2% glucose + 100 μM IAA and imaged every 3 minutes for 4 hours. Solid lines represent the average. Shaded areas represent 95% confidence intervals.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) FRAP analysis of Tem1 at dSPB in anaphase with or without depleting Cdc15. Cells of y3901 ( cdc15-AID , n = 6 cells) and y3900 ( cdc15-AID+OsTIR , n = 9 cells) were grown and imaged at room temperature in SC medium + 2% glucose + 100 μM IAA. Circles represent the average measurements of double normalized fluorescence intensities after correcting for photo-bleaching during acquisition. Solid lines are the average fit and shaded areas represent SD. (B) FRAP analysis of Tem1 at dSPB in anaphase with hydrolysis defective Bub2 mutant ( bub2R85A ). Cells of y2015 ( BUB2 , n = 6 cells) and y2971 ( bub2R85A , n = 6 cells) were grown and imaged at room temperature in SC medium + 2% glucose. Circles represent the average measurements of double normalized fluorescence intensities after correcting for photo-bleaching during acquisition. Solid lines are the average fit and shaded areas represent SD. (C) SPB localization of Tem1 in cells with defective GTP hydrolysis. Cells of y2015 ( WT , n = 69 cells), y2019 ( TEM1Q79L , n = 42 cells), and y2971 ( bub2R85A , n = 51 cells) were grown at 25 °C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the average. Shaded areas represent 95% confidence intervals.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Fluorescence, Mutagenesis

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) A simplified model for Tem1’s GTP/GDP cycle with or without the GAP. (B) Fraction of Tem1 GTP as predicted by the model (with GAP) as a function of the GAP-stimulated GTP hydrolysis rate k hyd , Tem1’s intrinsic nucleotide exchange rate k ex , Tem1’s intrinsic GTP hydrolysis rate k hyd int , dissociation constant for GAP binding Tem1 GTP K D T , dissociation constant for GAP binding Tem1 GDP K D D , or Tem1 concentration in the cell. Dashed lines represent the values used to make the predictions shown in A. (C) A simplified model for Tem1’s GTP/GDP cycle at dSPB in anaphase. The same model parameters were used as in A.

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

doi: 10.1101/2024.05.16.594582

Figure Lengend Snippet: (A) Quantification of Tem1 expression level. Whole-cell lysates of exponentially growing cells with different copy numbers of Tem1 integrated at the leu2 locus. Relative expression of Tem1 (indicated below) was quantified and normalized to the signal of Kar2. Underscored lanes represent strains used in this study: yA2587 (1xTem1, WT), y3693 (4xTem1), y3696 (9xTem1), y3692 (3xTem1), y3691 (2xTem1). (B) Predicted Tem1 GTP concentration as a function of Tem1 concentration in the cell (using the model illustrated in ). (C) SPB localization of Tem1 in diploid cells with two or one copy of yEGFP-TEM1 . Cells of y3713 (two copies, n = 20 cells) and y3711 (one copy, n = 26 cells) were grown at 25°C in SC medium + 2% glucose and imaged every 3 minutes for 4 hours. Solid lines represent the average. Shaded areas represent 95% confidence intervals. Note that the SPB localization profile of Tem1 heterozygous deletion (∼50% expression level) resembles the hypomorphic Tem1-GFP ( , ∼ 70% WT level).

Article Snippet: Three technical replicates of 18 µl MANT-GDP/GTP/GTPγS loaded Tem1 mixture were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics) at 30 °C.

Techniques: Expressing, Concentration Assay