Journal: Nature Communications

Article Title: Ubiquitination of CLIP-170 family protein restrains polarized growth upon DNA replication stress

doi: 10.1038/s41467-022-33311-y

Figure Lengend Snippet: a Tip1 ubiquitination is abolished in dma1Δ cells. 6His-Myc-tagged ubiquitin (6His-myc-Ub) was expressed from the P nmt1 promoter in wild-type or dma1Δ cells carrying Tip1−6HA and was pulled down with Ni 2+ -NTA beads. b Tip1 is most ubiquitinated in cells treated by hydroxyurea (HU). Yeast cells were arrested at different stages in the cell cycle using a drug HU (12 mM HU for 5.5 h) or various cell cycle mutants and then subjected to immunoblotting with Cdc2 as a loading control. c Deubiquitinating enzyme USP2 treatment removes the ladder of slow-migrating bands of Tip1−6HA. Cells grown in YE5S liquid medium were first treated with HU and then cell lysates were prepared and treated without or with USP2 before being subject to immunoprecipitation. d Tip1 ubiquitination is abolished in dma1 mutants. Domain architecture of Dma1 is shown. Asterisks indicate I194A and R64A mutations. Tip1 ubiquitination was detected by immunoblotting in wild-type or mts3-1 background cells with Cdc2 as a loading control. e Tip1 is more stable in dma1 mutants. Tip1−6HA was detected by immunoblotting in indicated strains with Cdc2 as a loading control. The relative protein level was quantified with a ratio between Tip1-6HA and Cdc2 in wild-type cells being set as 1.00. Bars represent mean ± SD from three independent replicates. Two-tailed unpaired t- test was used to derive p values. f The rate of Tip1 proteolysis in dma1 mutants is reduced. Time-course samples from cycloheximide (CHX)-treated cells were subject to immunoblotting. Relative Tip1-HA levels were quantified with a ratio between Tip1-6HA and Cdc2 at time 0 h being set as 1.00. Results are representative of two independent experiments. g Dma1 mediates ubiquitination of Tip1 via both K48 and K63 chain linkages. Strains expressing wild-type or different mutants of 6His-Myc-tagged ubiquitin as indicated were subjected to ubiquitination assay as in ( a ). The K48 and K63 mutants are ubiquitins with all Lys residues except K48 or K63 mutated to Arg, respectively, and K0 denotes ubiquitin with all 7 Lys residues mutated to Arg.

Article Snippet: To remove the potential ubiquitin or phosphorylation modifications of target proteins, cell lysates were incubated with recombinant human USP2 (R&D Systems, Cat. No. E-504) at a final concentration 50 nM for 2 h at 4 °C or 15 U of CIAP (ThermoFisher Scientific, Cat. No. 18009027) for 45 min at 37 °C, respectively, before being proceeded for immunoprecipitation.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Immunoprecipitation, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Ubiquitination of CLIP-170 family protein restrains polarized growth upon DNA replication stress

doi: 10.1038/s41467-022-33311-y

Figure Lengend Snippet: a Defective inhibition of bipolar growth in HU-treated dma1Δ . Cells of indicated genotypes grown in EMM5S liquid medium at 30 °C before or after being treated with 12 mM HU for 5.5 h were stained with calcofluor before scoring. Asterisks indicate bipolar cells. b Cell length is increased in dma1Δ cells upon defective DNA replication. Cells were either grown and treated with HU as in ( a ) or were cultured at 25 °C or shifted from 25 to 37 °C for 4 or 6 h (for pol1-1546 cells). Live cell length was measured. c DNA replication defects induced by HU or pol1-1546 mutation promote the accumulation of Dma1 at cell ends. Cells were grown and treated as in ( b ), cell images were captured after being fixed and fluorescence intensities of Dma1-mNeonGreen at cell ends were quantified. Asterisks indicate the representative cells enlarged in Supplementary Fig. . d , e dma1Δ is defective in inhibition of accumulation of cell-polarity landmarks CRIB and Tea4 upon defective DNA replication induced by HU. Cells of indicated genotypes were grown, treated, and imaged as in ( c ). Fluorescence intensities of GFP signals at cell ends were quantified. Representative cells are outlined and enlarged in Supplementary Fig. . f Binding affinity between Tea4 and Tip1 is elevated in dma1Δ cells and in wild-type cells treated with deubiquitinating enzyme USP2. Yeast lysates from indicated strains were treated with HU together with or without USP2 and incubated with bacteria-expressed MBP-Tea4 in in vitro pull-down assays. The relative levels of Tea4-bound Tip1 were quantified by comparing ratio of band intensities of Tip1-6HA from bead-bound and total lysate (WCE) with ratio in wild-type strain or USP2-untreated sample being set as 1.00. n = 3. Bars represent mean ± SD. For a – e , pooled data from three independent experiments are shown as mean ± SD; n indicates total cell numbers counted or measured for each strain; for a – f , p values were determined by two-tailed unpaired t- test. A.U. arbitrary units. Scale bars, 10 μm.

Article Snippet: To remove the potential ubiquitin or phosphorylation modifications of target proteins, cell lysates were incubated with recombinant human USP2 (R&D Systems, Cat. No. E-504) at a final concentration 50 nM for 2 h at 4 °C or 15 U of CIAP (ThermoFisher Scientific, Cat. No. 18009027) for 45 min at 37 °C, respectively, before being proceeded for immunoprecipitation.

Techniques: Inhibition, Staining, Cell Culture, Mutagenesis, Fluorescence, Binding Assay, Incubation, Bacteria, In Vitro, Two Tailed Test