Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

Palmitoylated Rho2 is targeted to the plasma membrane and is essential during morphogenesis but not for GTPase activity. (A) Deconvolved images of cells from strains LSM500 (GFP-Rho2-HA-CCIIS, control), LSM501 (GFP-Rho2-HA-SCIIS), LSM502 (GFP-Rho2-HA-CSIIS), and LSM503 (GFP-Rho2-HA-SSIIS) grown in YES medium and observed by fluorescence microscopy. GFP fluorescence density histograms across the longitudinal axis (dotted white lines) of representative cells in late G₂ phase are shown. (B) Density gradient centrifugation in 10 to 65% sucrose of cell extracts from growing cultures of strains LSM521 (GFP-Rho2-HA-CCIIS, SPAC1B2.03c-GFP, control) and LSM522 (GFP-Rho2-HA-SCIIS, SPAC1B2.03c-GFP). Aliquots from the indicated fractions (fraction 1 is the bottom of the tube) were subjected to Western blot analysis with anti-GFP antibody to detect GFP-Rho2 and SPAC1B2.03c-GFP (endoplasmic reticulum marker) fusions, anti-Pma1 antibody (plasma membrane marker), anti-Pep12 antibody (endosomal marker), and anti-ATP6V1B2 (Vma2) antibody (vacuole marker). (C) Strain MI700 (rho2Δ) was transformed separately with plasmid pREP3X-Rho2-HA-CCIIS (control), pREP3X-Rho2-HA-SCIIS, pREP3X-Rho2-HA-CSIIS, or pREP3X-Rho2-HA-SSIIS and thereafter grown for 18 h in the absence of thiamine (−B1). Cell morphology was analyzed by fluorescence microscopy after staining of cells with calcofluor white. (D) Serially diluted cells from transformants described for panel C were spotted on EMM2 plates with (+B1) or without (−B1) 5 mM thiamine and incubated for 4 days at 28°C. (E) Cell extracts from strains expressing genomic integrated versions of rho2⁺ alleles described for panel C were precipitated with GST-RBD and blotted against anti-HA antibody (top). Total HA-Rho2 was estimated by Western blotting with anti-HA antibody (middle), and Pmk1-HA was detected in the same extracts as the loading control (bottom). Quantification of the ratio of GTP bound to total Rho2 is shown.

In vivo palmitoylation of Rho2 is essential for signaling to the cell integrity pathway during vegetative growth and in response to stress. (A) Strains MI700 (rho2Δ), LSM400 (Rho2-HA-CCIIS; control [C]), LSM401 (Rho2-HA-SCIIS), LSM402 (Rho2-HA-CSIIS), and LSM403 (Rho2-HA-SSIIS) were grown in YES medium, and activated and total Pmk1s were detected with anti-phosho-p42/44 and anti-HA antibodies, respectively. (B) Strain MI102 (pmk1Δ) and those described for panel A were grown in YES medium, and serially diluted cells were spotted on YES plates supplemented with either 0.1 M or 0.2 M MgCl₂ plus 1 μg/ml FK506 (VIC). (C) Strains described for panel A were grown as above and treated with either 0.6 M KCl or 1 μg/ml caspofungin or subjected to a hypotonic treatment. (D) Serially diluted cells of strains described for panel B were spotted on YES plates supplemented with 0.8, 1, or 1.2 μg/ml caspofungin. (E) Strain MI700 (rho2Δ, Pmk1-HA) was transformed with plasmid pREP3X-Rho2-HA-CCIIS (control), pREP3X-Rho2-HA-SCIIS, pREP3X-Rho2-HA-CSIIS, or pREP3X-Rho2-HA-SSIIS, and the corresponding transformants were grown for 20 h in the presence or absence of thiamine (B1). Both activated and total Pmk1s were detected as previously described.

Artificial plasma membrane targeting bypasses the Rho2 need for palmitoylation in order to signal. (A) Deconvolved images of cells from strains LSM500 (GFP-Rho2-HA-CCIIS, control) and LSM970 (GFP-Rho2-HA-RitC). The percentage of GFP-Rho2 at the plasma membrane (PM) with respect to the whole cell is shown at the right (n ≥ 15 cells). (B) Density gradient centrifugation of cell extracts from cultures of strains LSM521 (GFP-Rho2-HA-CCIIS, SPAC1B2.03c-GFP, control) and LSM970 (GFP-Rho2-HA-RitC). Aliquots from the indicated fractions were subjected to Western blot analysis with anti-GFP and anti-Pma1 antibodies (plasma membrane marker). (C) Strains LSM400 (Rho2-HA-CCIIS, control), MI700 (rho2Δ), LSM403 (Rho2-HA-SSIIS), and LSM971 (Rho2-HA-RitC) were grown in YES medium, and activated and total Pmk1s were detected with anti-phosho-p42/44 and anti-HA antibodies, respectively. (D) Serially diluted cells of strains described for panel C were spotted on YES plates supplemented with either 0.05 M or 0.1 M MgCl₂ plus 1 μg/ml FK506 (VIC) or with 0.6 or 1 μg/ml caspofungin. (E) Strains LSM400 and LSM971 were treated with either 0.6 M KCl (left) or subjected to a hypotonic treatment (right). Activated and total Pmk1s were detected as previously shown.

Rho1 C-terminal polybasic sequence disturbs palmitoylation and signaling of geranylgeranylated but not farnesylated Rho2. (A) C-terminal sequences of Rho2 and Rho2-Rho1 chimeras. Rho1 C-terminal sequences are underlined, whereas amino acids derived from Rho2 terminal sequences are in bigger letters. (B) Deconvolved images of cells from strains LSM500 (GFP-Rho2-HA-CCIIS, control), LSM505 (GFP-Rho2-HA-RCILL), LSM506 (GFP-Rho2-HA-CCILL), and LSM507 (GFP-Rho2-HA-CCILS). The percentage of GFP-Rho2 at the plasma membrane with respect to the whole cell is shown at the right (n ≥ 15 cells; *, P < 0.05 in mutants compared to the control). (C) Strain MI700 (rho2Δ, Pmk1-HA) was transformed separately with plasmid pREP3X-Rho2-HA-CCIIS (control), pREP3X-Rho2-HA-RCILL, pREP3X-Rho2-HA-CCILL, or pREP3X-Rho2-HA-CCILS, and serially diluted cells from the respective cultures were spotted on EMM2 plates with (+B1) or without (−B1) 5 mM thiamine and incubated for 4 days at 28°C. (D) Strains LSM400 (Rho2-HA-CCIIS, control), LSM405 (Rho2-HA-RCILL), LSM406 (Rho2-HA-CCILL), LSM407 (Rho2-HA-CCILS), and MI700 (rho2Δ) were grown in YES medium, and both activated and total Pmk1s were detected as described for Fig. 1. (E) Strains described for panel D were grown in YES medium, and serially diluted cells were spotted on YES plates supplemented with 0.05 M or 0.1 M MgCl₂ plus 1 μg/ml FK506 (VIC). (F) Strains described for panel D were either treated with 0.6 M KCl or subjected to a hypotonic treatment. (G) Rho2 palmitoylation assayed by the acyl-biotinyl switch assay in cell lysates from strains described for panel D.

Rho GDI Rdi1 negatively regulates the cell integrity pathway through a Rho1-dependent mechanism. (A) Exponentially growing cultures of strains MI200 (control), MI700 (rho2Δ), LSM750 (rdi1Δ), and LSM760 (rho2Δ rdi1Δ), were grown in YES medium. Both activated and total Pmk1s were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. (B to D) Strains LSM400 (Rho2-HA-CCIIS, control) and LSM770 (Rho2-HA-CCIIS rdi1Δ) (B), LSM405 (Rho2-HA-RCILL) and LSM780 (Rho2-HA-RCILL rdi1Δ) (C), and LSM401 (Rho2-HA-SCIIS) and LSM790 (Rho2-HA-SCIIS rdi1Δ) (D) were treated with 0.6 M KCl. Aliquots were harvested at timed intervals, and activated and total Pmk1s were detected as described above.

Erf2 acyltransferase is the major protein responsible for Rho2 palmitoylation in vivo. (A) Exponentially growing cultures of strains MI200 (control), LSM810 (akr1Δ), LSM820 (pfa3Δ), LSM830 (pfa5Δ), LSM840 (erf2Δ), and LSM850 (erf4Δ) were treated with 0.6 M KCl. Aliquots were harvested at timed intervals, and activated and total Pmk1s were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. (B) Strains MI200 (control) and LSM840 (erf2Δ) were subjected to a hypotonic treatment, and both activated and total Pmk1s were detected. (C) Strains MI200 (control), LSM840 (erf2Δ), and LSM860 (erf2Δ akr1Δ pfa3Δ pfa5Δ) were subjected to hypotonic treatment, and both activated and total Pmk1s were detected. (D) Deconvolved images of cells from strains LSM500 (GFP-Rho2-HA-CCIIS, control) and LSM841 (GFP-Rho2-HA-CCIIS erf2Δ). The percentage of GFP-Rho2 at the plasma membrane with respect to the whole cell is shown at the right (n = 14 cells; *, P < 0.05 in erf2Δ cells compared to the control). (E) Rho2 palmitoylation assayed by the acyl-biotinyl switch assay (upper blot) in cell lysates from strains LSM400 (Rho2-HA-CCIIS, control), LSM842 (Rho2-HA-CCIIS erf2Δ), and LSM852 (Rho2-HA-CCIIS erf4Δ). (F) Exponentially growing cultures of strains MI700 (rho2Δ), LSM401 (Rho2-SCIIS), MI200 (control), LSM840 (erf2Δ), and LSM860 (erf2Δ akr1Δ pfa3Δ pfa5Δ) were grown in YES medium, and both activated and total Pmk1s were detected as above. (G) Serially diluted cells of strains described for panel F were spotted on YES plates supplemented with 0.05 M or 0.1 M MgCl₂ plus 1 μg/ml FK506 (VIC) and incubated for 3 days at 28°C.