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.
Citation: Fang Y, Jaiseng W, Ma Y, Hu L, Yamazaki S, Zhang X, et al. (2014) E3 Ubiquitin Ligase Pub1 Is implicated in Endocytosis of a GPI-Anchored Protein Ecm33 in Fission Yeast. PLoS ONE 9(1): e85238. doi:10.1371/journal.pone.0085238
Editor: Juan Mata, University of Cambridge, United Kingdom
Received: March 21, 2013; Accepted: November 30, 2013; Published: January 14, 2014
Copyright: © 2014 Fang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (No. 31071094) and Program for Liaoning Excellent Talents in University (No. LJQ2012067) to Yue Fang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Protein ubiquitylation, the process by which proteins are covalently modified by the small protein ubiquitin (Ub), is one of the most prevalent protein post-translational modifications in all eukaryotes from yeast to humans. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes such as receptor endocytosis, intracellular signaling, cell-cycle control, transcription, DNA repair, gene silencing, and stress response –. Aberrations in the ubiquitylation system have been implicated in the pathogenesis of major diseases such as cancer, diabetes, ion channel dysfunction, and neurodegenerative disorders , .
The ubiquitylation reactions were catalysed by a cascade of enzymes composed of a unique ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin-protein ligase (E3). Target proteins can be modified with a single Ub molecule on one (mono-ubiquitylation) or several (multi-monoubiquitylation) lysine residues. Alternatively, Ub molecules can be ligated to one another to form Ub chains where each monomer is linked to a lysine residue of previous Ub moiety (poly-ubiquitylation) , . Ub indeed harbors seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) all of which can be used for the attachment of another Ub . Mono-ubiquitylation provides a signaling mechanism that regulates important cellular pathways such as DNA repair, histone function, and endocytosis –, and K48-linked poly-ubiquitylation provides an important recognition signal for degradation in the proteasome . Moreover, K6- and K63-linked poly-ubiquitylation serves non-proteasomal functions in various signaling and trafficking pathways –. There is a subfamily of genes that encode different ubiquitin conjugating enzymes. On the other hand, ubiquitin ligases are more varied, depending on their structures. A combination of specialized ubiquitin-conjugating enzymes and ubiquitin ligases is responsible for highly specific recognition of the target proteins .
In budding yeast Saccharomyces cerevisiae, one of the best studied ubiquitin ligases is Rsp5p which belongs to the Nedd4 family. Rsp5p is involved in regulation of a broad array of cellular processes including endocytosis, multivesicular body (MVB) sorting, RNA export, transcription, lipid biosynthesis, mitochondrial inheritance, and protein catabolism via mono- and poly-ubiquitinate target proteins , , . In fission yeast Schizosaccharomyces pombe, there are three ubiquitin ligase Nedd4/Rsp5 homologues, namely pub1+, pub2+, and pub3+, which are HECT-type ubiquitin ligases . It has been shown that Pub1 is required for cells to tolerate low pH conditions , to regulate leucine uptake in response to the presence of NH4+ , and to participate in cell cycle control , . Discovering new functions of highly homologous ligases, such as Pub1 in fission yeast, may provide useful information which can be easily utilized in the deciphering of similar process in higher eukaryotes.
In our previous study, we identified a mutant allele of the cis4+ gene that encodes a zinc transporter belonging to the cation diffusion facilitator (CDF) protein family, and we characterized the role of Cis4 in Golgi membrane trafficking in fission yeast . More recently, we screened for multicopy suppressors of the MgCl2-sensitive phenotype of the cis4-1 mutant and identified three genes encoding GPI-anchored proteins, namely Ecm33, Aah3, and Gaz2 . In this study, we further screened for multicopy suppressors of the phenotypes of the cis4-1 mutant, and identified two genes, ubi1+ and ubc4+. The ubi1+ gene, encoding an N-terminal ubiquitin fused to the ribosomal protein L40, belongs to the class of ubiquitin genes whose translation product is ubiquitin-ribosome fusion protein Ubi1. The ubc4+ gene, encoding a ubiquitin conjugating enzyme Ubc4 that is essential for cell growth, is required for mitotic transition and regulating the nuclear protein quality , . Upon further investigation of the pathway requiring Ubc4, we found that overexpression of Ubi1 or Ubc4 failed to suppress the phenotypes of the double deletion of cis4+ and pub1+ genes. In addition, we showed that at exponential phase GFP-Ecm33 localized at the cell surface and the medial region in wild-type cells. In particular, during the post-exponential and stationary phase, GFP-Ecm33 in wild-type cells was internalized and mostly localized to the Golgi/endosomes, whereas in Δpub1 cells, it was still stably localized at the plasma membrane. Taken together, these results strongly suggested that the function of Ubc4 involving in suppressing the phenotypes of Δcis4 occurred in Pub1-dependent manner. Furthermore, our results demonstrate that Pub1 is implicated in endocytosis of a GPI-anchored protein Ecm33 and regulation of cell wall integrity in fission yeast.
Isolation of the ubi1+ and ubc4+ genes as multicopy suppressors of zinc transporter cis4-1 mutant
We have previously demonstrated that zinc transporter Cis4 plays a role in Golgi membrane trafficking in fission yeast . Recently, we screened for multicopy suppressors of the MgCl2-sensitive phenotype of the cis4-1 mutant and identified three genes encoding GPI-anchored proteins, namely Ecm33, Aah3, and Gaz2 . In order to identify novel genes that are involved in Cis4 function, we further screened for genes that when overexpressed could suppress the MgCl2 sensitivity of cis4-1 mutant. As shown in Figure 1A, the cis4-1 mutant cells grew well in rich YPD medium, however, in the presence of 0.15 M MgCl2 the cis4-1 cells failed to grow whereas wild-type cells grew well. Notably, when the ubi1+ and ubc4+ genes, respectively, were overexpressed, the cis4-1 mutant cells grew in the presence of 0.15 M MgCl2 (Figure 1A). Then we examined in Δcis4 mutants the effects of the overexpression of ubi1+ and ubc4+ genes, respectively, and results showed that both genes also suppressed the MgCl2-sensitive growth defect of the Δcis4 cells (our unpublished data).
(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.
Recently, we reported that the Δecm33 cells exhibited similar cis phenotype including FK506 sensitivity and MgCl2 sensitivity to that of the Δcis4 cells . Then, we examined whether overexpression of ubi1+ or ubc4+ suppress the phenotypes of Δecm33 cells, and results showed that overexpression of both genes suppressed the phenotypes of the Δecm33 cells (Figure 1B). Thus, together with previous results, our study suggests that the phenotypes of Δcis4 and Δecm33 mutants are overlapped, which might be due to the involvement of Cis4 and Ecm33 in the regulation of cell wall integrity , .
Next, we investigated the effect of other genes, that is, ubi3+ encoding a ubiquitin-ribosome fusion protein, ubi4+ encoding a multiubiquitin, rhp6+ and ubc16+ encoding ubiquitin conjugation enzymes, respectively. The Δcis4 mutants transformed with these genes were tested for growth on YPD containing 0.12 M MgCl2. The results showed that overexpression of ubi3+ or ubi4+ gene slightly but significantly suppressed the phenotypes of the Δcis4 mutant (Figure 1C). We also investigated the effects of the overexpression of ubi3+ or ubi4+ in Δecm33 mutants, and results showed that overexpression of both genes failed to suppress the MgCl2-sensitive growth defect of the Δecm33 cells (Figure 1C). On the other hand, overexpression of both rhp6+ and ubc16+ genes failed to suppress the phenotypes of the Δcis4 mutants, clearly indicating that this property is highly specific to Ubc4 (our unpublished data).
Effects of K6R, K11R, K48R, and K63R Ubi1 mutation on the suppression of the phenotypes of Δcis4 cells
Many studies have demonstrated that K48-linked poly-ubiquitylation are usually associated with proteasomal degradation , whereas K6- and K63-linked chains serve non-proteasomal functions in various signaling and trafficking pathways –. To determine which configuration of the ubiquitin linkages is responsible for the complementation of Δcis4 phenotypes, we evaluated the effects of K6R, K11R, K48R, and K63R Ubi1 mutation on the suppression of the phenotypes of Δcis4 cells. These mutant proteins have the invariant lysine in position 6, 11, 48, or 63 mutated to arginine, respectively, and expression of these mutants has a chain-terminating effect, resulting in the premature termination of ubiquitin chain. As shown in Figure 2A, all these mutants except K63R mutant could fully suppress the MgCl2- and FK506-sensitive phenotypes of Δcis4 cells, suggesting that K6-, K11-, or K48-linked poly-ubiquitylation is not involved in the suppression of the phenotypes of Δcis4 mutant. To exclude the possibility that overexpression of K63R Ubi1 causes an irrelevant growth inhibitory defect, we tested the effect of overexpression of Ubi1 and K63R Ubi1 in wild-type cells in plates containing the MgCl2 or FK506 as a control. The results showed that wild-type cells overexpressing K63R Ubi1 grew normal similar to that of overexpressing Ubi1 in the presence of MgCl2 or FK506 (Figure 2B). These results suggest that K63-linked poly-ubiquitylation is involved in this cellular process. We also examined the effects of these mutant proteins on the phenotypes of Δecm33 cells, and the results showed that all of them exhibited similar genetic suppression profile of the Δecm33 cells as compared to that of the Δcis4 cells (Figure 2C).
(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.
Genetic interaction between cis4+ and pub1+ genes
In a recent study, Stoll et al. demonstrated that the essential redundant function performed by Ubc4p and Ubc5p is with Rsp5p, the only essential HECT-type E3 in budding yeast . This raised the possibility that Ubc4 may serve as an E2 functioning together with Rsp5p homologues, namely Pub1, Pub2, or Pub3 in fission yeast. For this purpose, we first constructed the Δcis4Δpub1, Δcis4Δpub2, and Δcis4Δpub3 double deletion mutants. As shown in Figure 3A, 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 (Figure 3A). Similar to the Δcis4Δpub1 cells, the Δcis4Δpub3 cells showed less sensitive to MgCl2 than that of the Δcis4 mutants. On the other hand, the Δcis4Δpub2 cells exhibited the similar MgCl2-sensitive phenotype as compared with that of Δcis4 mutants (Figure 3A). These results suggested that there is a strong genetic interaction between Cis4 and Pub1, or Pub3, but not Pub2. Consistently, the amino acid sequence similarity between Pub1 and Pub2 is relatively low, whereas Pub1 and Pub3 are with a higher amino acid identity .
(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.
Furthermore, we investigated the effect of Ubc4 or Ubi1 overexpression on the phenotypes of these double mutants. Most strikingly, overexpression of Ubc4 or Ubi1 failed to suppress the MgCl2-sensitive phenotype of the Δcis4Δpub1 mutants, but could still suppress the phenotype of the Δcis4Δpub2 and Δcis4Δpub3 cells (Figure 3B). These results suggest that the suppression of Δcis4 by ubc4+ or ubi1+ overexpression requires Pub1. However, it remains unclear why the MgCl2 sensitivity of Δcis4 is alleviated by Pub1 mutation (Figure 3A). The MgCl2-sensitivity reflects the dynamic equilibrium among multiple cellular activities in terms of its impact on cell wall integrity. Our findings suggest that Pub1 as well as Pub1-mediated ubiquitylation may be involved in several cellular activities that play antagonistic roles in the regulation of cell wall integrity.
The Δpub1 mutants showed pleiotropic phenotypes related to cell wall integrity
In order to gain insight into the function of Pub1 in fission yeast, we tested the phenotypes of the Δpub1 mutants in greater detail. As shown in Figure 4A, in addition to high and cold temperature sensitivity, the Δpub1 cells exhibited hypersensitivity to immunosuppressant drug FK506 and micafungin, a (1, 3)-β-D-glucan synthase inhibitor, like other cell wall integrity deficient mutants such as Δcis4 . Moreover, the Δpub1 cells also showed marked hypersensitivity to antifungal drug clotrimazole, and some metals including CaCl2, CdCl2, ZnSO4, and LiCl2. Defects in cell wall integrity can sometimes be compensated for by increases in the osmolarity of the growth media . So we examined whether these phenotypes of the Δpub1 mutants were suppressible by osmotic stabilization of the medium with sorbitol. Notably, addition of 1.2 M sorbitol suppressed all of the phenotypes except ZnSO4 sensitivity and LiCl2 sensitivity (Figure 4B). We further analyzed whether Δpub1 mutants show altered resistance to cell wall damaging agents such as β-glucanase (Zymolyase). The β-glucanase treatments on the wild-type cells and Δpmk1 cells, which lack a MAP kinase regulating the cell wall integrity of fission yeast  were also performed as negative control and positive control, respectively. Interestingly, the results showed that the sensitivity of the Δpub1 mutants to β-glucanase was not distinctly different from that of the wild-type cells, although the Δpmk1 cells showed more marked sensitivity to β-glucanase (Figure 4C). We also examined the phenotypes of Δpub2 and Δpub3 cells, and the results showed that no significant differences were detected between wild-type and these mutants under conditions as described above (our unpublished data).
(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.
Enhanced CDRE transcriptional activity and Ecm33 promoter activity in Δpub1 cells
We previously established an in vivo real-time monitoring system of calcineurin activity utilizing the reporter harboring the calcineurin-dependent response element (CDRE)-fused to luciferase, and showed that high extracellular CaCl2 concentration and cell wall damaging agents caused an increase in the CDRE-reporter activity in fission yeast . The above result that the Δpub1 cells exhibited hypersensitivity to FK506, a calcineurin inhibitor, led us to investigate whether Pub1 deletion affect the CDRE reporter activity due to its defective cell wall. Results showed that the Δpub1 mutants displayed markedly enhanced CDRE reporter response in the absence or presence of 0.1 M CaCl2, compared with that of the wild-type cells (Figure 4D).
We also analyzed the Ecm33 promoter activity in Δpub1 mutants using the reporter construct containing the 0.5 kb DNA fragment of the ecm33+ gene promoter region-fused to luciferase (Materials and Methods). Takada et al reported that the reporter activity of Ecm33 promoter could be enhanced in response to a variety of stimuli including CaCl2 . Our results showed that the Δpub1 mutants exhibited significantly enhanced Ecm33 promoter activity in the absence or presence of 0.2 M CaCl2, compared with that of the wild-type cells (Figure 4E).
Localization of GFP-Ecm33 in Δpub1 cells
As mentioned above, overexpression of ubi1+ as well as ubc4+ genes suppressed the MgCl2 sensitivity of the Δecm33 cells. This prompted us to hypothesize that ubiquitylation may play roles in membrane trafficking of the GPI-anchored proteins. Then, we examined whether Pub1 deletion affect the localization of GFP-Ecm33. Previously, we have reported that GFP-Ecm33 localized at the growing cell tips of the cell surface and the medial regions in the wild-type cells of exponential phase growth . Here, we examined the localization of GFP-Ecm33 in the wild-type cells during different stages of growth. As shown in Figure 5A, in wild-type cells, GFP-Ecm33 clearly localized at the growing cell tips of the cell surface or the medial regions at exponential phase. When cells were further grown for 12 hours to post-exponential phase, GFP-Ecm33 primarily localized as dot-like structures that were observed in the cytoplasm as well as at the cell surface and the division site. When cells were further grown for 36 hours to stationary phase, GFP-Ecm33 mostly localized at dot-like structures in the cytoplasm rather than at the cell surface (Figure 5A). To determine whether the dot-like fluorescence of GFP-Ecm33 indicate the Golgi/endosome-associated localization, we examined the co-localization of GFP-Ecm33 with the endocytic tracer dye FM4–64 during an early stage of endocytosis. After 5 min of dye uptake, most of the GFP-Ecm33 dot-like structures co-localized with FM4–64-positive structures (Figure 5A), suggesting that GFP-Ecm33 localized to the Golgi/endosome compartments in the wild-type cells at post-exponential and stationary phase. These are consistent with the idea that GPI-anchored proteins were endocytosed and transported to the recycling endosomes via a compartment named GPI-anchored proteins enriched early endosomal compartments (GEECs), followed by recycling back to the plasma membrane , .
(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.
Next, we observed the localization of GFP-Ecm33 in Δpub1 cells. Intriguingly, the results showed that GFP-Ecm33 was observed throughout the whole cell surface and the division site in the Δpub1 cells at exponential phase (Figure 5B). Notably, GFP-Ecm33 was still stably localized at the plasma membrane in Δpub1 cells at post-exponential and stationary phase (Figure 5B). We also examined the localization of GFP-Ecm33 in Δubi1, ubc4-P61S, Δpub2, and Δpub3 cells. Results showed that in Δubi1 cells GFP-Ecm33 localized at the cell surface and the medial regions at exponential and post-exponential phase. When cells were further grown to stationary phase, GFP-Ecm33 localized as intracellular dot-like structures in addition to the cell surface, suggesting that in Δubi1 cells GFP-Ecm33 was endocytosed more slowly than that in wild-type cells (Figure 5B). In ubc4-P61S mutant, GFP-Ecm33 localized at the cell surface and the division site through all the stages of growth, similar to that in the Δpub1 cells (Figure 5B). On the other hand, the localization of GFP-Ecm33 in Δpub2 and Δpub3 cells was not distinctly different from that observed in wild-type cells at all stages of growth (Figure 5B). Altogether, our results demonstrated that Pub1, Ubc4 as well as Ubi1 are implicated in endocytosis of Ecm33.
To determine whether Ecm33 is ubiquitinated by ubiquitin ligase Pub1, GFP-Ecm33 and GST-ubiquitin were co-expressed in wild-type and Δpub1 cells, and cells were analyzed by the pull-down assay. Aat1, a protein known to be ubiquitinated by Pub1, was also tested as a positive control in this experiment. Protein extracts were prepared from cells incubated for 24 hours. At that time point, more than half of the Ecm33 were endocytosed into the cytoplasm. As shown in Figure 5C, results showed that equivalent amount of GST-ubiquitin-bound proteins were recovered by glutathione beads from wild type strains expressing either Aat1-GFP or GFP-Ecm33, but only the Aat1-GFP could be easily detected in the purified fractions in the examined condition. The reason is unknown why GST-ubiquitin-bound proteins were poorly recovered from the Δpub1 mutant. Three bands were detected in the lysate containing GFP-Ecm33. Probably, the lower one is GFP-Ecm33, whereas the upper two are GPI anchor-linked GFP-Ecm33.
Previously, we have reported that several membrane trafficking mutants such as Δapm1 cells showed abnormal localization of GFP-Ecm33 . The apm1+ gene encodes µ1A subunit of the clathrin-associated adaptor protein complex 1(AP-1) implicated in Golgi/endosome function . In order to analyze the functional relationship between Pub1 and Apm1, we performed tetrad analysis by crossing Δpub1 with Δapm1, and constructed the Δpub1Δapm1 double mutants. As expectedly, the results showed that the Δpub1Δapm1 mutants displayed more marked temperature sensitivity and MgCl2 sensitivity than that of their single mutants (Figure 5D), suggesting that there is a strong genetic interaction between Pub1 and Apm1. As described previously, the Δapm1 and Δcis4 cells showed abnormal localization of GFP-Ecm33 . Then we tested whether the Ecm33 localization in these mutants was altered in the absence of Pub1. Results showed that in Δapm1 cells GFP-Ecm33 localized as intracellular dot-like structures in addition to the cell surface at exponential and post-exponential phase, whereas GFP-Ecm33 mostly localized as intracellular dot-like structures at stationary phase. In contrast, in Δpub1Δapm1 cells, GFP-Ecm33 localized at the cell surface and the division site through all the stages of growth, similar to that in the Δpub1 cells (Figure 5E). On the other hand, in Δcis4 cells, localization of GFP-Ecm33 was similar to that in wild-type cells at exponential and post-exponential phase. However, GFP-Ecm33 localized to the intracellular dots and to the structure surrounding the nuclei that are considered to be the endoplasmic reticulum (ER) at stationary phase, similar to that in the zinc-deficient medium . Surprisingly, the Δpub1Δcis4 cells exhibited similar localization of GFP-Ecm33 as compared with that of Δcis4 cells through all the stages of growth (Figure 5E).
We further investigated whether Ubi1 or Ubc4 overexpression rescue the phenotypes of Δapm1 cells. As shown in Figure 5F, overexpression of ubi1+ and ubc4+ suppressed the MgCl2 sensitivity of Δapm1 cells, and overexpression of ubi1+ suppressed the temperature sensitivity of Δapm1 cells. However, overexpression of both genes failed to suppress these phenotypes of Δapm1 cells when Pub1 was deleted (Figure 5F). We also examined whether the abnormal localization of GFP-Ecm33 in the Δapm1 cells was changed by overexpressing Ubi1 or Ubc4. Results showed that GFP-Ecm33 localized on the cell surface or Golgi/endosomes in Δapm1 cells at exponential phase as previously described (Figure 5G, Δapm1 + vector). Notably, GFP-Ecm33 was mostly visible at the cell surface in Δapm1 cells that harbored ubi1+ (Figure 5G, Δapm1 + ubi1+), whereas GFP-Ecm33 was observed at the cell surface and Golgi/endosomes in Δapm1 cells that harbored ubc4+ (Figure 5G, Δapm1 + ubc4+). These results suggested that Ubi1 overexpression recovered normal Ecm33 localization, but Ubc4 overexpression could not. These data indicate again that Apm1 as well as Pub1-mediated ubiquitylation are implicated in membrane trafficking of GPI-anchored protein Ecm33.
In order to investigate whether involvement of Pub1 in the internalization of GPI-anchored protein is specific for Ecm33, we examined the localization of GFP-Gaz2, another GPI-anchored protein. As shown in Figure 5H, in wild-type cells, GFP-Gaz2 clearly localized at the cell surface or the medial regions at exponential phase. When cells were further grown to post-exponential phase and stationary phase, GFP-Gaz2 primarily localized as intracellular dot-like structures, similar to that of GFP-Ecm33. On the other hand, in Δpub1 cells, GFP-Gaz2 was observed at the cell surface and the division site at exponential phase. Notably, GFP-Gaz2 was still stably localized at the plasma membrane in Δpub1 cells at post-exponential and stationary phase, indicating that Gaz2 also internalizes in a Pub1-dependent manner (Figure 5H).
In the present study, we report that Pub1 participates in endocytosis of a GPI-anchored protein Ecm33 in fission yeast. Our findings also support the notion that Pub1 is implicated in regulation of cell wall integrity. To our knowledge, this is the first report of the involvement of an ubiquitin ligase in regulating the trafficking of GPI-anchored proteins.
Pub1 facilitates the endocytosis of a GPI-anchored protein Ecm33
An important finding of this study is the role of E3 ubiquitin ligase Pub1 in the endocytosis of GPI-anchored protein Ecm33. GPI-anchored proteins are mainly found on the plasma membrane. Accumulating evidence supports the idea that like other cell surface proteins, once GPI-anchored proteins have reached the plasma membrane, they are then subject to internalization, down-regulation and degradation. Ubiquitylation, one of the most common post-translational modifications, is required for degradation and endocytosis of transmembrane surface proteins . However, it remains unclear whether ubiquitylation is required for endocytosis of GPI-anchored proteins. It has been reported that in many cell types the main fraction of GPI-anchored proteins is delivered to GEECs, from which these proteins eventually reach recycling endosomes. The recycling rates of GPI-anchored proteins from the recycling endosomes are at least threefold to fourfold slower than other recycling membrane compartments. Alternatively, GEECs are trafficked to the late endosomes , . Consistently, here we showed that in wild-type cells GFP-Ecm33 localized to cell surface at exponential phase of growth, whereas it localized to the Golgi/endosomes at steady state. However, it should be noted that, in the Δpub1 cells and ubc4-P61S mutant, GFP-Ecm33 localized to the cell surface at all the stages of growth, strongly suggesting that Pub1 deletion affected the endocytosis of Ecm33 in fission yeast. Moreover, in Δubi1 GFP-Ecm33 was endocytosed more slowly than that in the wild-type cells. Our results presented here strongly suggest that ubiquitylation is implicated in endocytosis of Ecm33. However, Ecm33 is not ubiquitylated. The exact target of ubiquitylation is still unclear at the present. Nakase et al reported that Pub1-mediated ubiquitylation is required for localization and regulation of the Aat1 permease in fission yeast . In this study, our observations indicated that GPI-anchored protein Ecm33 was endocytosed in a Pub1-dependent manner (Figure 6) that is also required for the trafficking of non-GPI-anchored proteins in fission yeast. Ecm33 is important for cell wall function and involved in the negative feedback regulation of Pmk1 cell wall integrity signaling . As described in the results, MgCl2 sensitivity in Δcis4 cells is partially suppressed by pub1 mutation (Figure 3A). It is possible that cell surface localization of Ecm33 enhances the cell wall function when Pub1 deleted, thereby partially complemented the MgCl2 sensitivity of the Δcis4 cells. The exact physiological meaning of Ecm33 internalization is still unknown. Probably, internalization of Ecm33 serves as a critical signal for its involvement in the Pmk1 MAPK cell signaling. Given the high similarity between fission yeast and mammalian cells, this study may provide a basis for understanding the precise mechanism of endocytosis of GPI-anchored proteins in higher eukaryotes.
E2 ubiquitin-conjugating enzyme Ubc4 is involved in the cellular process related to Cis4
In general, specificity of ubiquitylation is afforded by specialized E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases, which recognize the target proteins with a high degree of selectivity. In budding yeast, Ubc4p functions with many E3 including Rsp5p, the anaphase-promoting complex, Pex10p, SCF (Skp1/cullin/F-box), and Not4p . However, in fission yeast, the identity of E3s with Ubc4 (E2) function is largely unknown. In a recent study, it was reported that Ubc4 and San1 (E3) are required for regulating nuclear protein quality in fission yeast . In the present study, our results showed that Ubi1 and Ubc4 overexpression suppressed the phenotypes of Δcis4 mutants, but did not suppress the phenotypes of the Δcis4Δpub1 mutants. Similarly, Ubi1 and Ubc4 overexpression suppressed the MgCl2-sensitive phenotype of Δapm1 mutants, but did not suppress the phenotype of the Δapm1Δpub1 mutants. These results suggest that Ubc4 may serve as an E2 functioning with Pub1 in the cellular processes related to Cis4 in fission yeast. We cannot formally exclude that an additional partner E3 may function with Ubc4 in suppression phenotypes of Δcis4 mutants, because Pub1 overexpression could not suppress the phenotypes of the Δcis4 mutants (our unpublished data). Furthermore, the results presented in this study support the notion that K6, K11, or K48-linked poly-ubiquitylation is not involved in the suppression of the Δcis4 mutants. It is possible that Ubc4 as well as Pub1 may be involved in the suppression of the Δcis4 mutants by mediating K63-linked poly-ubiquitylation.
Pub1 plays a role in cell wall integrity
Here, we present several lines of evidence that suggest a novel role of Pub1 in the cell wall integrity. First, the Δpub1 cells displayed hypersensitivity to a cell wall-damaging agent, micafungin that inhibits (1, 3)-β-d-glucan synthase essential for cell wall synthesis. Second, high osmolarity suppressed most of the phenotypes of Δpub1 cells. Third, the Δpub1 cells displayed hypersensitivity to FK506, a specific inhibitor of calcineurin that plays important roles in the regulation of cell wall integrity , . Moreover, the Δpub1 cells showed high CDRE-reporter activity that reflects an enhanced calcineurin activity probably caused by its defective cell wall. Also, the Δpub1 cells exhibited significantly enhanced Ecm33 promoter activity. Consistent with our results, Takada et al reported that Ecm33 is involved in Pmk1 MAPK-mediated cell wall integrity signaling . On the other hand, sensitivity to cell wall degrading enzyme β-glucanase of the Δpub1 mutant was not altered compared with that of wild-type cells. This atypical phenotype suggests that the cell wall structure composed of β-glucan might not be affected by Pub1 deletion. However, the yeast cell wall has a layered structure and is composed not only of glucan but also of mannoproteins as well as chitin. Therefore, our results indicate that the sensitivity to β-glucanase is not the sole criterion for assessment of defects in cell wall integrity. Thus, our findings demonstrate that Pub1 is involved in the regulation of the cell wall integrity, and cell wall integrity is one of the causes for the pleiotropic phenotypes of Δpub1. Consistently, it has been reported that cell wall is defective in rsp5–13 mutant . Probably, as shown in the present study, Pub1 deletion affects the function of various GPI-anchored proteins, such as Ecm33, that are essential for cell wall organization and cell viability, thereby causes the cell wall weakness. Likewise, it is possible that overexpression of ubi1+ and ubc4+ enhanced Pub1-involved cell wall integrity, thereby complemented the phenotypes of Δcis4 mutants.
Thus, although Ecm33 is not directly ubiquitylated, our present results suggest that Ubi1, Ubc4, and Pub1 play roles for the endocytosis of Ecm33. Cis4 and Apm1 are also involved in the membrane trafficking of Ecm33. All the deletion mutants studied in this study, that is, Δcis4, Δecm33, Δapm1, and Δpub1, showed defects in cell wall integrity and overexpression of ubi1+ or ubc4+ gene effectively suppressed the defects of Δcis4, Δecm33 as well as Δapm1 mutants (Figure 1A, 1B and Figure 5F). However, the MgCl2 sensitivity in the Δcis4 strain is apparently alleviated by Δpub1 mutation (Figure 3A) that may have an opposite effect of overexpression of ubi1+ or ubc4+. These findings suggest that Ecm33, Cis4, and Apm1 are involved in multiple molecular processes related to cell wall integrity and Ubi1-Ubc4-Pub1-mediated ubiquitylation play roles for the regulation of these molecular processes. Further studies are needed to more completely elucidate the molecular relationship among Cis4, Apm1, Ecm33 and these ubiquitin-related factors.
Materials and methods
Strains, media, genetic and molecular biology techniques
S. pombe strains used in this study are listed in Table 1. The complete medium, YPD, and the minimal medium, EMM, have been described previously . Standard S. pombe genetic and recombinant-DNA methods were performed as described previously except where noted . Gene disruptions are denoted by lowercase letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, pub1::ura4+). Gene disruptions are abbreviated by the gene preceded by Δ (for example, Δpub1). Proteins are denoted by roman letters and only the first letter is capitalized (for example, Pub1). Tacrolimus (FK506) was obtained from Astellas Pharma (Tokyo, Japan). All other chemicals and reagents were purchased from commercial sources.
Multicopy suppressor screen
The multicopy suppressor screen was performed as previously described . Briefly, a genomic library cloned into the vector pDB248  was transformed into the cis4-1 mutant. The Leu+ transformants were replica-plated onto YPD plates containing 0.15 M MgCl2 and the plasmid DNA was recovered from transformants that showed a plasmid-dependent rescue. These plasmids were then sequenced, revealing insertions containing the ubi1+ and ubc4+ genes, respectively, in addition to other multicopy suppressors as described previously .
Site-directed mutagenesis and generation of truncated Ubi1 mutants
Lys-to-Arg substitution at Lys6, Lys11, Lys48, and Lys63 form of the Ubi1 proteins were constructed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene). In the PCR amplification reaction, the primers used were summarized in Table 2. The amplified products containing these genes were digested with XhoI/BamHI, and the resulting fragments were subcloned into Blue-Script SK (+).
Knockout of the pub1+, pub2+, and pub3+ genes
The pub1+, pub2+, and pub3+ gene deletions with a genetic background of h+ leu1-32 ura4-D18 ade6-M210 or M216 were purchased from BioNEER (South Korea) . We constructed these gene deletion cells that were not auxotrophic for uracil or adenine by the genetic cross between wild-type cells HM123 and the above strains to make KP4766, KP6087, and KP6088 (Table 1).
Calcineurin-dependent Response Element (CDRE)-dependent reporter assay
The cells were transformed with the reporter plasmid (3×CDRE::luc (R2.2))  and were untreated or treated with 0.1 M CaCl2. The CDRE transcriptional activity was measured as described previously  with minor modifications. Briefly, the culture was diluted with fresh medium and was grown for further 3 hours at 27°C. Then, the culture was diluted to OD660 = 0.2, and was mixed with 0.5 mM D-luciferin. Aliquots of the cell culture were pipetted into a 96-well plate, and CaCl2 was added. EMM was used as a control. The mixture was incubated at 27°C for 3 hours, and light emission levels expressed as relative light units were measured using a new type of luminometer (AB-2350; ATTO Co.).
The ecm33+ promoter assay
The firefly luciferase reporter assay vector (pKB5721) was constructed as described previously . A 0.5-kb DNA fragment in the 5′flanking region of the ecm33+ gene was amplified by PCR (forward primer 4614, 5′-AA CTG CAG CAA GCT CCT CGT TGG TGT TGT GGCC-3′; reverse primer 4615, 5′-CCG CTC GAG ATT GAC TTT AGA CTA TAT AAT G-3′) and subcloned into the PstI/XhoI site of pKB5721.
Cells transformed with the above reporter plasmid were cultured at 27°C in EMM to midlog phase. The ecm33+ promoter activity was measured as described by Deng et al  with minor modifications. Briefly, the culture was diluted with fresh medium and was grown for further 3 hours at 27°C. Then, the culture was diluted to OD660 = 0.3, and was mixed with 0.5 mM D-luciferin. Aliquots of the cell culture were pipetted into a 96-well plate, and CaCl2 was added to a final volume and concentration of 100 μl and 200 mM, respectively. EMM was used as a control. The mixture was incubated at 27°C for 3 hours, and light emission levels expressed as relative light units were measured using luminometer (AB-2350; ATTO Co.).
Database searches were performed using the National Center for Biotechnology Information BLAST network service (www.ncbi.nlm.nih.gov) and the Sanger Center S. pombe database search service (www.sanger.ac.uk).
Microscopy and miscellaneous methods
Methods in light microscopy, such as fluorescence microscopy that was used to observe the localization of GFP-tagged proteins and FM4–64 labeling, were performed as described , . Tetrad analysis and GST pull-down assay were performed as previously described , .
We thank Susie O. Sio for critical reading of the manuscript. Original strain of Δpub1 cells (h− leu1-32 ura4-D18 pub1::ura4+) was provided by the National Bio-Resource Project (NBRP) of the MEXT, Japan. The mutant ubc4-P61S was a generous gift from Dr Hiroaki Seino.
Conceived and designed the experiments: YF TK. Performed the experiments: YF WJ LH SY XZ TH LS. Analyzed the data: YF WJ YM TK. Contributed reagents/materials/analysis tools: YF TK. Wrote the paper: YF TK.
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