Figure 7.
Several hairpin proteins behave as Get3 clients in yeast. (A) Fluorescence microscopy images of WT and ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Images are representative of three biological replicates with >100 cells imaged for each replicate. Scale bar: 2 µM. (B) Fluorescence microscopy images of ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Cells were stained with MitoTracker Orange (MitoTracker) to visualize mitochondria. Images are representative of three biological replicates with >100 cells imaged for each replicate. Arrows indicate co-localization. Scale bar: 2 µM. (C) Schematic representation of the topology of proteins visualized in A. The number of amino acid residues present N- or C-terminally of the predicted hairpins are indicated for each protein. (D) Immunoblot of cell lysates from WT and ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Pgk1 served as a loading control. Images are representative of three biological replicates. (E) Quantification of D. Bars represent the average of three biological replicates with individual data points shown as gray dots. Error bars indicate standard deviation of the mean. The P values calculated using the two-sided Welch’s t test are shown with numbers and represented as follows: ** < 0.01; *** < 0.001. (F) Fluorescence microscopy images of WT and ∆get3 strains expressing GAL1 promoter-driven N-terminally GFP-tagged Lam1, Sip3, Prm9, Tsc10 ectopically and Ubx2 genomically. Strains were grown in synthetic dropout media containing 2% raffinose and imaged 1 h after transition to media containing 2% galactose (Gal). Images are representative of three biological replicates with >200 cells imaged for each replicate. Scale bar: 2 µM. (G) Fluorescence microscopy images of ∆get3 strains expressing genomically C-terminally mTagBFP2-tagged Hsp104 and GAL1 promoter-driven N-terminally GFP-tagged Lam1, Sip3, Prm9 ectopically. Strains were grown in synthetic dropout media containing 2% raffinose and imaged 1 h after transition to media containing 2% galactose (Gal). Images are representative of three biological replicates with >200 cells imaged for each replicate. Arrows indicate co-localization. Scale bar: 2 µM. Source data are available for this figure: SourceData F7.

Several hairpin proteins behave as Get3 clients in yeast. (A) Fluorescence microscopy images of WT and ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Images are representative of three biological replicates with >100 cells imaged for each replicate. Scale bar: 2 µM. (B) Fluorescence microscopy images of ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Cells were stained with MitoTracker Orange (MitoTracker) to visualize mitochondria. Images are representative of three biological replicates with >100 cells imaged for each replicate. Arrows indicate co-localization. Scale bar: 2 µM. (C) Schematic representation of the topology of proteins visualized in A. The number of amino acid residues present N- or C-terminally of the predicted hairpins are indicated for each protein. (D) Immunoblot of cell lysates from WT and ∆get3 strains expressing the indicated genomically N-terminally GFP-tagged proteins under the control of the NOP1 promoter (NOP1pr). Pgk1 served as a loading control. Images are representative of three biological replicates. (E) Quantification of D. Bars represent the average of three biological replicates with individual data points shown as gray dots. Error bars indicate standard deviation of the mean. The P values calculated using the two-sided Welch’s t test are shown with numbers and represented as follows: ** < 0.01; *** < 0.001. (F) Fluorescence microscopy images of WT and ∆get3 strains expressing GAL1 promoter-driven N-terminally GFP-tagged Lam1, Sip3, Prm9, Tsc10 ectopically and Ubx2 genomically. Strains were grown in synthetic dropout media containing 2% raffinose and imaged 1 h after transition to media containing 2% galactose (Gal). Images are representative of three biological replicates with >200 cells imaged for each replicate. Scale bar: 2 µM. (G) Fluorescence microscopy images of ∆get3 strains expressing genomically C-terminally mTagBFP2-tagged Hsp104 and GAL1 promoter-driven N-terminally GFP-tagged Lam1, Sip3, Prm9 ectopically. Strains were grown in synthetic dropout media containing 2% raffinose and imaged 1 h after transition to media containing 2% galactose (Gal). Images are representative of three biological replicates with >200 cells imaged for each replicate. Arrows indicate co-localization. Scale bar: 2 µM. Source data are available for this figure: SourceData F7.

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