Fig. 7
From: Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies
Cytosolically expressed VHH1SMT3 and VHH2SMT3 compromise yeast growth. (A) Smt3p overexpressed under the control of the CUP1 promoter in EBY100 yeast is readily detected by VHH1SMT3. Left: whole cell lysate of EBY100 yeast transformed with His7-Smt3p plasmid, as indicated. Shown is an immunoblot of overexpressed His7- Smt3p upon induction with 100 µM CuSO4 detected by anti-His antibody (middle) and VHH1SMT3 (right). (B) Anti-HA tag immunoblot to detect expression of VHH1SMT3, VHH2SMT3 and VHH1SMT3 equipped with a C terminal nuclear localization tag (NLS), induced in response to varying concentrations (0, 100, 300 µM) of CuSO4. (C) Anti-HA tag immunoblot of GFP- and NLS-tagged versions of VHH1SMT3 in yeast. (D) Yeast spot dilution growth assay with the VHH constructs shown in (B,C) on CuSO4-containing (0, 100, 300 µM) yeast synthetic media agar plates. The four constructs showing a significant growth defect with increasing concentration of CuSO4 are shown on the right. (E) Fluorescence microscopy showing EBY100 yeast cells expressing VHH1SMT3-GFP tagged versions with and without an NLS. Images for phase-contrast (left), the GFP channel (middle) and an overlay of both (right) are shown. Notice increased nuclear localization of VHH1SMT3 with an NLS tag, regardless of whether the NLS is located at the N or C terminus.
