Fig. 5: Targeted deubiquitination of Tip1 suppresses Tip1 hyper-ubiquitination and promotes cell polar growth.

a–c Fusing a deubiquitinating enzyme module of UL36 in cis with Tip1 suppresses Tip1 hyper-ubiquitination and promotes cell polar growth. a Schematic diagram of UL36 module (15-260aa) fusion with Tip1 ectopically expressed under the P_adh11 promoter and its potential enzymatic activity on Tip1 deubiquitination. b, c Cells were grown and treated with HU as in Fig. 4a and then Tip1−6HA-UL36(15-260aa) was detected by immunoblotting (b) and cell length was measured (c). The C40S mutation renders a catalytically dead version of UL36 module. d–h Targeted deubiquitination of Tip1 in trans based on the abscisic acid (ABA)-induced specific PYL and ABI interaction also suppresses Tip1 hyper-ubiquitination and promotes cell polar growth. d Scheme of the design of the Tip1-PYL and Ubp7-ABI constructs. Tip1 was fused with 3HA and the PYL ___domain (residues 33-209) of the ABA receptor regulatory component PYL1, and the catalytic ___domain (residues 201-875) of fission yeast deubiquitinating enzyme Ubp7 was fused with GFP and the ABI ___domain (residues 126-423) of ABI1. ABA-induced PYL-ABI dimerization may enable Ubp7 to approach and deubiquitinate Tip1. ub, ubiquitin. e, f Ubp7-GFP-ABI fusion protein is sufficiently recruited to cell tips by Tip1-PYL in the presence of ABA. Both Tip1−3HA-PYL and Ubp7-GFP-ABI were ectopically expressed in tip1Δ strain. Cells were grown as in Fig. 4a, then treated with 12 mM HU or 250 μM ABA separately or simultaneously for 5.5 h. Images of fixed cells were captured (e) and the percentages of cells with Ubp7-GFP-ABI signals at cell ends were quantified (f). g, h Ubp7(201-875aa)-GFP-ABI1(126-423aa) suppresses Tip1−3xHA-PYL1(33-209aa) hyper-ubiquitination and promotes cell polar growth. Cells were grown and treated as in (e) and then Tip1-3xHA-PYL1(33-209aa) was detected by immunoblotting (g) and cell length was measured (h). Asterisks indicate cell tips enlarged in insets. For c, f, and h, pooled data from three independent experiments are shown as mean ± SD; n indicates total cell numbers counted or measured for each strain; p values were determined by two-tailed unpaired t-test. Scale bar, 10 μm.