E3 ubiquitin ligase Pub1 is implicated in endocytosis of a GPI-anchored protein Ecm33 in fission yeast

Abstract

We previously identified three glycosylphosphatidylinositol (GPI)-anchored proteins including Ecm33, as multicopy suppressors of the phenotypes of a mutant allele of cis4(+) that encodes a zinc transporter in fission yeast. Here, we further identified two multicopy suppressor genes, ubi1 (+) and ubc4 (+), encoding ubiquitin-ribosomal fusion protein and ubiquitin conjugating enzyme E2, respectively. In addition, Ubi1 or Ubc4 overexpression failed to suppress the phenotypes of the double deletion of cis4 (+) and pub1 (+) gene, which encodes a HECT-type ubiquitin ligase E3. During exponential phase GFP-Ecm33 localized at the growing cell tips of the cell surface and the medial region in wild-type cells. Notably, during the post-exponential and stationary phase, GFP-Ecm33 in wild-type cells was internalized and mostly localized to the Golgi/endosomes, but it was still stably localized at the cell surface in Δpub1 cells. The Δpub1 cells showed osomoremedial phenotypes to various drugs indicating their defects in cell wall integrity. Altogether, our findings reveal a novel role for Pub1 in endocytosis of Ecm33 and regulation of cell wall integrity in fission yeast.

Figure 1. Isolation of Ubi1 and Ubc4 as multicopy suppressors of the cis4-1 mutant cells.

(A) The cis4-1 mutant cells were transformed with either the pDB248 multicopy vector, or the vector containing ubi1 ⁺ or ubc4 ⁺. Cells were then streaked onto plates containing YPD, or YPD plus 0.15 M MgCl₂, and then incubated for 4 days at 30°C. (B) The Δecm33 cells were transformed with either the pDB248 multicopy vector, or the vector containing ubi1 ⁺ or ubc4 ⁺. Cells were then streaked onto plates containing YPD, or YPD plus 0.15 M MgCl₂, and then incubated for 4 days at 30°C. (C) The Δecm33 and Δcis4 cells were transformed with either the pDB248 multicopy vector, or the vector containing ubi3 ⁺ or ubi4 ⁺. Cells were then spotted onto plates containing YPD, or YPD plus 0.12 M MgCl₂, and then incubated for 4 days at 30°C.

Figure 2. K6-, K11-, or K48- linked poly-ubiquitylation is not involved in the suppression of the phenotypes of Δcis4 mutant.

(A) The Δcis4 cells harboring the vector for the indicated proteins were spotted onto plates containing YPD, YPD plus 0.15 M MgCl₂, or YPD plus 0.5 μg/ml FK506, and then incubated for 4 days at 30°C. (B) Wild-type cells harboring the vector for the indicated proteins were spotted onto plates containing YPD, YPD plus 0.15 M MgCl₂, or YPD plus 0.5 μg/ml FK506, and then incubated for 4 days at 30°C. (C) The Δecm33 cells harboring the vector for the indicated proteins were streaked onto plates containing YPD, YPD plus 0.15 M MgCl₂, or YPD plus 0.5 μg/ml FK506, and then incubated for 4 days at 30°C.

Figure 3. Genetic interaction between cis4 ⁺ and pub1 ⁺ genes.

(A) The Δcis4Δpub1 mutants were more markedly sensitive to high and cold temperature than that of the Δpub1 mutants, but less sensitive to MgCl₂ than that of the Δcis4 mutants. Wild-type cells, Δcis4, Δpub1, Δpub1Δcis4, Δpub2, Δpub2Δcis4, Δpub3, and Δpub3Δcis4 cells were spotted onto each plate as indicated, and then incubated at 30°C for 4 days, at 36°C for 3 days or at 17°C for 13 days. (B) Overexpression of Ubc4 or Ubi1 failed to suppress the MgCl₂-sensitive phenotype of the Δcis4Δpub1 mutants, but could suppress the phenotypes of the Δcis4Δpub2 and Δcis4Δpub3 cells. Wild-type cells, Δpub1Δcis4, Δpub2Δcis4, or Δpub3Δcis4 cells transformed with a control vector or the vector containing ubi1 ⁺ and ubc4 ⁺ respectively, were spotted onto YPD, or YPD plus MgCl₂ plates, and then incubated at 30°C for 4 days.

Figure 4. The Δpub1 mutants showed defects in cell wall integrity.

(A) Phenotypes of the Δpub1 cells. Wild-type cells and Δpub1 cells were streaked onto each plate as indicated, and then incubated at 30°C for 4 days. (B) High osmolarity suppressed all of the phenotypes of the Δpub1 cells except ZnSO₄ sensitivity and LiCl₂ sensitivity. Wild-type cells and Δpub1 cells were streaked onto each plate as indicated, and then incubated at 30°C for 4 days, at 37°C for 3 days or at 17°C for 13 days. (C) Cell wall digestion of the Δpub1 cells and wild-type cells by β-glucanase. Cells exponentially growing in YPD medium were harvested and incubated with β-glucanase (Zymolyase) at 30°C and subjected to vigorous shaking. Cell lysis was monitored by the measurement of the optical density at 660 nm. The data shown are representative of multiple experiments. (D) The Δpub1 cells displayed an enhanced calcineurin activity. Wild-type cells and Δpub1 cells harboring the multicopy plasmid (3×CDRE::luc (R2.2) reporter vector were grown to exponential phase in liquid EMM at 30°C, and the reporter activity was monitored as described in Materials and Methods. The data shown are representative of multiple experiments. (E) The Δpub1 mutants showed significantly enhanced Ecm33 promoter activity. Wild-type cells and Δpub1 cells harboring the multicopy plasmid Ecm33 promoter reporter vector were grown to exponential phase in liquid EMM at 30°C, and the reporter activity was monitored as described in Materials and Methods.

Figure 5. Localization of GFP-Ecm33 in the Δpub1 cells.

(A) In wild-type cells, GFP-Ecm33 clearly localized at the growing cell tips of the cell surface or the medial regions at exponential phase, whereas it primarily localized as dot-like structures in the cytoplasm at post-exponential and stationary phases. Wild-type cells expressing chromosome-borne GFP-Ecm33 were cultured to exponential phase, and further grown for 12 hours to post-exponential phase, and for 36 hours to stationary phase in EMM medium at 30°C, and were examined by fluorescence microscopy, respectively (Materials and Methods). Wild-type cells expressing chromosome-borne GFP-Ecm33 cultured in EMM medium at post-exponential and stationary phases were incubated with FM4–64 fluorescent dye for 5 min at 27°C to visualize Golgi/endosomes. GFP-Ecm33 localization and FM4-64 fluorescence were examined under a fluorescence microscope. Bar: 10 μm. (B) The Δpub1, Δpub2, Δpub3, Δubi1 and ubc4-P61S mutants expressing chromosome-borne GFP-Ecm33 were cultured in EMM medium at 30°C as described in Figure 5A, and were examined by fluorescence microscopy. Bar: 10 μm. (C) In cells expressing GFP-Ecm33 from the chromosomally integrated gene, GST-ubiquitin was expressed from the harboring plasmid at 27°C. GST-tagged ubiquitin was pulled down by glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies. Tubulin was used as a control to show the presence of equal amount of proteins in each lane and was immunoblotted using anti-Tubulin antibody. (D) Genetic interaction between Pub1 and Apm1. The Δpub1Δapm1 double mutants showed more marked temperature sensitivity than their single mutants. Wild-type, Δpub1, Δapm1, and Δpub1Δapm1 cells were spotted onto YPD, or YPD plus MgCl₂ plates, and then incubated for 4 days at the temperatures as indicated. (E) The Δapm1, Δpub1Δapm1, Δcis4, and Δpub1Δcis4 mutants expressing chromosome-borne GFP-Ecm33 were cultured in EMM medium at 30°C as described in Figure 5A, and were examined by fluorescence microscopy. Bar: 10 μm. (F) Ubi1 or Ubc4 overexpression suppressed the MgCl₂-sensitive phenotype of the Δapm1 mutants, whereas failed to suppress the phenotype of Δpub1Δapm1 mutants. Wild-type, Δapm1, or Δpub1Δapm1 cells transformed with a control vector, ubi1 ⁺, or ubc4 ⁺, were spotted onto YPD, or YPD plus MgCl₂ plates, and then incubated for 4 days at the temperatures as indicated. (G) Ubi1 suppressed the defective localization of GFP-Ecm33 in Δapm1 cells. Wild-type and Δapm1 cells expressing chromosome-borne GFP-Ecm33 cells transformed with pDB248 or the vector containing ubi1 ⁺ and ubc4 ⁺ were cultured in YPD medium at 27°C. The GFP-Ecm33 localization was examined under the fluorescence microscope. Bar, 10 mm. (H) Cells transformed with the vector expressing GFP-Gaz2 were cultured in EMM medium at 30°C as described in Figure 5A, and were examined by fluorescence microscopy. Bar: 10 μm.

Figure 6. Cartoon illustrates molecular mechanisms of membrane trafficking of GPI-anchored protein Ecm33.

Schematic diagram is based on the collective findings in this report. The block arrows indicate effects. A question mark indicates an unknown target of the ubiquitylation.