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¹ Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
² Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka, 577-8502, Japan
³ Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan
⁴ RIKEN Plant Science Center, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Kanagawa, Japan
⁵ Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1⁺ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1⁺ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
Most of the newly synthesized secretory proteins are translocated into the endoplasmic reticulum (ER) and then transported to the plasma membrane via the Golgi by intracellular vesicle trafficking. The vesicle trafficking requires a mechanism to direct vesicles to their specific target compartments. The Rab/Ypt family of Ras-related monomeric GTPases localizes at different intracellular compartments, and defines the specificity between a vesicle and its target during the early docking events in various steps of the exocytic pathway (Pfeffer 1999).

We have been studying membrane trafficking in fission yeast because this system is amenable to genetic analysis and has many advantages in terms of its relevance to higher systems. To identify components that are functionally related to calcineurin signaling, we developed a genetic screen that utilized a specific inhibitor of calcineurin, the immunosuppressant drug FK506, and have searched for its mutations that display immunosuppressant- and temperature-sensitive phenotypes. By this genetic screen, we have identified the Rab/Ypt family small GTPase Ypt3/Its5. Our previous results demonstrated that Ypt3 is essential for cell viability and is required for the exit from the trans-Golgi as well as for the later step of the exocytic pathway, suggesting that Ypt3 plays the roles of both Ypt31/32 and Sec4 in the secretory pathway of the budding yeast (Cheng et al. 2002). In the ypt3-i5 gene, a single base change (G to A) caused the replacement of the conserved Arg29 in Ypt3 protein by a histidine residue. GFP-Ypt3R29H mutant protein no longer localized at discrete regions of the cell and instead, the whole cytoplasm and the nucleus were stained, indicating that the conserved Arg29 is primarily required for the specific localization of Ypt3 protein (Cheng et al. 2002).

Using the same genetic screen, here we report the identification of another GTPase mutant its6-1/ryh1-i6, an allele of the ryh1⁺ gene encoding a homolog of mammalian Rab6 and budding yeast Ypt6 GTPase. In mammalian cells, Rab6 GTPase appears to function in intra-Golgi transport (Martinez et al. 1994, 1997) and COPI/ARF1-independent retrograde transport from the Golgi to the ER (Girod et al. 1999; White et al. 1999). In budding yeast, Ypt6 has been implicated in having a role in retrograde membrane trafficking from endosomes to the trans-Golgi network (Tsukada & Gallwitz 1996; Tsukada et al. 1999; Bensen et al. 2001; Siniossoglou & Pelham 2001) or in anterograde (Li & Warner 1996, 1998) and retrograde (Bensen et al. 2001) Golgi transport.

In fission yeast, Ryh1 was first characterized by Hengst et al. (1990) whose study showed that the ryh1 null mutants were unable to grow at high temperature. In the same study, the invertase of ryh1 cells was properly secreted but had a faster electrophoretic mobility compared to that of wild-type cells, and that the temperature-sensitive phenotype of ryh1 cells was complemented by the expression of the human Rab6 cDNA. In another study by Aiba et al. (1998) a high-osmolarity-sensitive mutant was isolated and the ryh1 mutant was shown to be severely sterile. Although the function of this member of the Rab6/Ypt6/Ryh1 subfamily protein has been well studied, still its exact role in the secretory pathway has not yet been completely defined. In the present study, we show that Ryh1 localized at the Golgi/endosome and that the ryh1-i6 mutant exhibited phenotypes similar to ypt6 mutants, indicating that Ryh1 is implicated in retrograde traffic from endosome to the Golgi.

Furthermore, we showed for the first time the genetic and functional interaction between two Rab GTPases Ryh1 and Ypt3. Firstly, ryh1⁺ gene deletion was synthetically lethal with ypt3-i5 mutation. Secondly, both the ryh1-i6 and ypt3-i5 mutants exhibited the phenotypes such as defective acid phosphatase secretion and cell wall integrity in addition to their its phenotypes. Thirdly, over-expression of GDP-conformational mutant, Ryh1T25 N inhibited the growth of ypt3-i5 mutant and exacerbated the phenotypes of ypt3-i5 mutant, while it had no effect on the wild-type cells. Fourthly, GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome. The localizations of Ryh1 and Ypt3 in the ypt3-i5 and ryh1-i6 mutant cells are distinct from those in the wild-type cells. Together, our results suggest Ryh1 plays a role in the secretory pathway and may have a potential overlapping function with Ypt3.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Isolation of the its6-1 mutant

We have developed a simple genetic screen using FK506, a specific inhibitor of calcineurin, and have thus far identified several genes that function in membrane trafficking. The first gene identified in our previous work was ypt3⁺/its5⁺ gene that encodes a homolog of mammalian Rab11 and budding yeast Ypt31 and Ypt32 (Cheng et al. 2002). The second gene isolated was apm1⁺/cis1⁺ gene that encodes a homolog of the mammalian µ1 A subunit of the clathrin-associated adaptor protein-1 complex (Kita et al. 2004). A new addition to this series of genes isolated by a genetic screen that functionally interacts with calcineurin in membrane trafficking is the its6⁺/ryh1⁺ gene. As shown in Fig. 1A, its6-1 mutants grew equally well as compared with the wild-type cells at 27 °C. However, its6-1 mutant cells could not grow at 36 °C nor could they grow on YPD plate containing FK506 at 27 °C, whereas wild-type cells grew normally (Fig. 1A).

View larger version (77K): [in this window] [in a new window]   Figure 1  Mutation in the its6+/ryh1+ gene causes immunosuppressant- and temperature-sensitive phenotypes. (A) The immunosuppressant and temperature sensitivities of the its6-1/ryh1-1 mutant cells and its6/ryh1-deleted (ryh1) cells. Cells transformed with the multicopy vector pDB248 or the vector containing the ryh1+ gene were streaked onto each plate containing YPD, YPD plus 0.5 µg/mL FK506, then incubated for 4 days at 27 °C or 3 days at 36 °C, respectively. (B) Comparison of amino acid sequences among fission yeast Ryh1, corresponding region of human Rab6A and the budding yeast Ypt6. Sequence alignment was performed using the Clustal W program. Asterisks indicate the identical amino acids. Arrow indicates the mutation site in the Gln136 of Ryh1, which when mutated to a stop codon resulted in the immunosuppressant- and temperature-sensitive phenotype.

The its6⁺ gene was cloned by complementation of the temperature-sensitive growth defect (Fig. 1A). Nucleotide sequencing of the cloned DNA fragment revealed that its6⁺ gene is identical to the ryh1⁺ gene (SPAC4C5.02c) (Hengst et al. 1990) which encodes the Ryh1 protein of 201 amino acids that is very similar to the mammalian Rab6 GTPase (109/169, 64% identity) and to S. cerevisiae Ypt6 (136/210, 64% identity) (Fig. 1B). Linkage analysis was performed (see Experimental procedures) and results indicated the allelism between the ryh1⁺ gene and the its6-1 mutation. Accordingly, we renamed the its6-1 mutant as ryh1-i6 mutant.

As shown in Fig. 1A, FK506-sensitive growth defect of the its6-1/ryh1-i6 mutant was also suppressed by ryh1⁺ gene. To determine the mutation site, genomic DNA from ryh1-i6 mutant was isolated, and the full-length coding region of the ryh1-i6 gene was sequenced. The C to T nucleotide substitution caused a glutamine to be altered to a termination codon at the amino acid position 136, and resulted in a truncated protein product lacking 65 amino acids downstream of the mutation (Fig. 1B).

The ryh1 cells also showed temperature sensitivity at 36 °C and FK506 sensitivity at 27 °C (Fig. 1A, ryh1+vector).

Genetic interaction between two Rab family proteins Ryh1 and Ypt3

As both the ypt3-i5 and ryh1-i6 mutants showed its (immunosuppressant- and temperature-sensitive) phenotype, and as both are members of the Rab/Ypt small GTP-binding protein family, we further examined the genetic interaction between ryh1⁺ and ypt3⁺ genes. First, we performed tetrad analysis by crossing ypt3-i5 mutant with ryh1 (Fig. 2A). Results showed that no double mutant was obtained, indicating that ypt3-i5 mutation and ryh1⁺ deletion are synthetically lethal.

View larger version (109K): [in this window] [in a new window]   Figure 2  The genetic interaction between ryh1+ and ypt3+ gene. (A) The ryh1 deletion is synthetically lethal with the ypt3-i5 mutation. Tetrad analysis of progeny derived from crossing KP2580 (h–leu1-32 ura4-D18 ryh1::ura4+) with KP659 (h+leu1-32 his2 ura4-D18 ypt3-i5). Colonies grown on YPD at 27 °C were streaked onto various plates as indicated. Colonies that failed to grow on YPD at 36 °C, and on YPD with FK506 were predicted to be ypt3-i5, and those that grew in the absence of uracil (EMM +Leu +His) were predicted to be ryh1. The rest of the colonies were predicted to be wild-type cells. Spores that failed to grow were predicted to be ryh1 ypt3-i5 double mutants. Arrows and white rectangle indicate tetrads of non-parental ditype. (B) The ypt3-i5 mutants were hypersensitive to the expression of the dominant-negative Ryh1T25N mutant. The dominant-negative Ryh1T25N mutant was expressed in wild-type (wt) and ryh1-i6 mutant cells, and cells were streaked onto EMM plate with or without 4 µM thiamine then incubated for 4 days at 27 °C. (C) Over-expression of the GTPase-deficient mutant Ryh1Q70L caused a growth defect on wild-type cells but had no effect on the growth of the ypt3-i5 mutant cells. Wild-type cells (wt) and ypt3-i5 mutant cells transformed with pREP1-ryh1+, or pREP1-ryh1Q70L were streaked onto EMM plate with or without 4 µM thiamine then incubated for 4 days at 27 °C. (D) The pleiotropic phenotypes of ryh1-i6 mutant cells were differentially suppressed by the over-expression of Ryh1Q70L and Ykt6. Serial ten-fold dilutions starting with OD660 = 0.3 of log-phase wild-type cells (wt), and ryh1-i6 mutant cells carrying an empty multicopy vector or vector containing the genes as indicated were spotted (5 µL) onto YPD plates with or without the indicated additives. Thiamine contained in YPD medium attenuates the expression from the nmt1 promoter. Plates were incubated for 4 days at 27 °C or 3 days at 36 °C and photographed. (E) The over-expression of Ykt6 had no effect on the temperature- or FK506-sensitive phenotypes of ypt3-i5 mutant cells. Cells were spotted as described above.

Ryh1Q70L mutant protein and Ykt6 differentially suppressed the pleiotropic phenotypes of ryh1-i6 mutant cells

Furthermore, we constructed a mutationally activated form of the Ryh1 protein to represent the GTP conformational mutant. Here, the conserved Gln70 among Rab6/Ryh1 protein was mutated to Leu (Ryh1Q70L) and this mutation was constructed by analogy to the GTPase-deficient mutant Rab6Q72L (Martinez et al. 1994). Expression of the dominant-active mutant is expected to affect the kinetics of transport related to its wild-type counterpart. Ryh1Q70L and wild-type Ryh1 were over-expressed using pREP1 vector (Basi et al. 1993) in wild-type and ypt3-i5 mutant cells. Notably, over-expression of Ryh1Q70L, but not that of the wild-type Ryh1, caused the growth defect of the wild-type cells (Fig. 2C). On the other hand, over-expression of Ryh1Q70L or wild-type Ryh1 had no effect on the growth of the ypt3-i5 mutant cells (Fig. 2C).

As described above, the nmt1 promoter-driven over-expression of Ryh1Q70L mutant inhibited the growth of the wild-type cells but not that of ypt3-i5 mutant cells. To further characterize the Ryh1Q70L mutant protein, we examined whether the attenuated over-expression of Ryh1Q70L could suppress the pleiotropic phenotypes of the ryh1-i6 mutant. Since the rich YPD medium contains thiamine, the nmt1 promoter-driven over-expression of Ryh1 or Ryh1Q70L should be considerably attenuated. As shown in Fig. 2D, the attenuated over-expression of Ryh1Q70L suppressed the FK506 and CaCl₂ sensitivity, but contrary to our expectation, it failed to suppress the temperature sensitivity of the ryh1-i6 mutant.

In the process of gene cloning, we isolated the prenylated SNARE ykt6⁺ gene (Sogaard et al. 1994; McNew et al. 1997) as a multicopy suppressor of the temperature-sensitive phenotype of the ryh1-i6 mutant (see Experimental procedures). In contrast to the results obtained with Ryh1Q70L, the ykt6⁺ gene when over-expressed, partially suppressed the temperature-sensitive growth defect of the ryh1-i6 mutant cells, but it failed to suppress the FK506 or CaCl₂ sensitivity of the ryh1-i6 mutants (Fig. 2D). Over-expression of ykt6⁺ gene had no effect on the growth of the ypt3-15 mutant cells (Fig. 2E).

Intracellular localization of Ryh1 and Ypt3

The genetic and phenotypic analyses suggest that Ryh1 and Ypt3 may functionally interact in membrane trafficking. Then, we studied the intracellular localization of Ryh1 and Ypt3 by tagging the 5' end of ryh1⁺ and ypt3⁺ with the sequence encoding GFP. GFP-Ryh1 is fully functional and its expression complemented both the temperature-sensitive and immunosuppressant-sensitive growth defects of the ryh1 cells (Fig. 3A). GFP-Ryh1Q70L also showed identical complementation ability as compared with that of Ryh1Q70L (Figs 2D and 3A).

View larger version (179K): [in this window] [in a new window]   Figure 3  Intracellular localization of GFP-Ryh1 and GFP-Ypt3. (A) GFP-tagged Ryh1 and Ryh1Q70L are functionally similar to non-tagged proteins. Cells transformed with the multicopy vector pREP1-GFP-Ryh1, pREP1-GFP-Ryh1Q70L or the empty vector were streaked onto each plate containing YPD, YPD plus 0.5 µg/mL FK506, or YPD plus 0.1 M CaCl2, then incubated for 4 days at 27 °C or 3 days at 36 °C, respectively. (B) Subcellular localization of Ryh1 tagged with GFP in wild-type cells. The fused gene encoding GFP-Ryh1, GFP-Ryh1Q70L or GFP-Ryh1-i6 were integrated into the chromosome of wild-type cells under the control of the nmt1 promoter of pREP1 (see Experimental procedures). Cells were grown to early log phase in EMM containing 4 µM thiamine, then incubated with FM4-64 dye for 5 min to visualize Golgi/endosomes. Arrowheads indicate the dot-like structures of GFP-Ryh1 that correspond to Golgi/endosomes stained with FM4-64. Double arrowheads indicate the dot-like structures stained with FM4-64 that did not co-localize with GFP-Ryh1Q70L. Arrows indicate the localization of GFP-Ryh1Q70L associated with plasma membrane. Bar, 10 µm. (C) Co-localization of Ryh1 and Krp1. Wild-type cells expressing GFP-Ryh1 or GFP-Ryh1Q70L were transformed with the pREP1 vector expressing Krp1 fused to RFP at its C-terminus. Cells were grown to early log phase in EMM containing 4 µM thiamine. Bar, 10 µm. (D) Immunoblot analysis of GFP-Ryh1, GFP-Ryh1-i6 and GFP-Ryh1Q70L. Wild-type cells expressing GFP-tagged proteins were cultured in EMM containing 4 µM thiamine at 27 °C. The respective proteins were separated by SDS-PAGE and analyzed by immunoblotting using anti-GFP antiserum. Endogenous Cdc4 was used as a loading control and was immunoblotted using anti-Cdc4 antiserum. (E) Subcellular localization of GFP-Ypt3. Arrowheads indicate the dot-like structures of GFP-Ypt3 that correspond to Golgi/endosomes stained with FM4-64. Double arrowheads indicate dot-like structures stained with FM4-64, but did not co-localize with GFP-Ypt3. Arrows indicate GFP-Ypt3 at the cell tips. Bar, 10 µm. (F) Subcellular localization of GFP-Ryh1 in ypt3-i5 mutant cells. Arrowheads indicate the dot-like structures of GFP-Ryh1 that correspond to Golgi/endosomes stained with FM4-64. Double arrowheads indicate the dot-like structures of GFP-Ryh1 that were not stained with FM4-64. Bar, 10 µm. (G) Subcellular localization of GFP-Ypt3 in ryh1-i6 mutant cells. Arrowheads indicate the dot-like structures of GFP-Ypt3 that correspond to Golgi/endosomes stained with FM4-64. Bar, 10 µm.

We then examined the co-localization of GFP-Ryh1 with Krp1 fused to monomeric red fluorescent protein (RFP) at its C-terminus. Krp1 is a furin/Kex2 homolog that resides in the Golgi (Powner & Davey 1998). As shown in Fig. 3C, GFP-Ryh1 mostly co-localized with Krp1-RFP, consistent with their localization at the Golgi. Consistent with the microscopic obervation as shown in Fig. 3B, immunoblot analysis showed that the protein level of GFP-Ryh1-i6 was very low suggesting the instability of the protein. However, the comparable stability of GFP-Ryh1Q70L and GFP-Ryh1, as shown by the almost identical protein level, suggest that the inability of Ryh1Q70L to suppress the temperature sensitivity of the ryh1-i6 mutant is not due to an indirect effect of the protein's instability (Fig. 3D).

We also examined the co-localization of GFP-Ypt3 with FM4-64 (Fig. 3E). As described in our previous report (Cheng et al. 2002) and as shown here, GFP-Ypt3 localized at the cell tips (Fig. 3E, arrows) and less prominently at the intracellular dot-like structures. However, the GFP fluorescence at the cell tips occasionally co-localized with FM4-64 at an early stage of endocytosis, suggesting that a part of Ypt3 also localizes at Golgi/endosome compartments (Fig. 3E, arrowheads). But a considerable portion of the FM4-64 fluorescent dots in the cytosol did not co-localize with GFP-Ypt3 (Fig. 3E, double arrowheads).

In conclusion, GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations.

Localization of GFP-Ryh1 in ypt3-i5 mutant and GFP-Ypt3 in ryh1-i6 mutant

To further study the interaction between Ryh1 and Ypt3, we examined the localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant. In wild-type cells, most of the GFP-Ryh1 dot-like fluorescence co-localized with FM4-64 at an early stage of endocytosis (Fig. 3B). In contrast, in ypt3-i5 mutant cells, a considerable part of GFP-Ryh1 localized at dot-like structures that did not co-localize with FM4-64 (Fig. 3F, double arrowheads). On the other hand, in wild-type cells, a considerable part of GFP-Ypt3 did not co-localize with FM4-64 (Fig. 3E). In contrast, in ryh1-i6 mutant cells most of the GFP-Ypt3 dots, though not all, co-localized with FM4-64 (Fig. 3G). Interestingly, the localization pattern of GFP-Ypt3 in ryh1-i6 mutant is similar to that of GFP-Ryh1 in the wild-type cells (Fig. 3B).

Ryh1 is required for the intra-Golgi trafficking

As Ryh1 localized at the Golgi/endosome, we first checked the acid phosphatase glycosylation in ryh1-i6 mutant cells to identify whether Ryh1 function in the intra-Golgi trafficking. On native gels, acid phosphatase isolated from ryh1-i6 cells harboring vector migrated significantly faster than that from the wild-type cells (Fig. 4A). On the other hand, acid phosphatase isolated from ypt3-i5 cells harboring vector migrated similarly as that from the wild-type cells. Notably, acid phosphatase isolated from ypt3-i5 cells over-expressing the dominant-negative Ryh1T25N mutant protein migrated significantly faster than those from wild-type cells or the wild-type cells over-expressing the dominant-negative mutant protein (Fig. 4A). In contrast to the ypt3-i5 mutant, the dominant-negative mutant protein Ryh1T25N had no effect on the migration speed of acid phosphatase isolated from other its or ypt mutants (data not shown). These results suggest that Ryh1 is involved in the trafficking pathways required for normal glycosylation (presumably intra-Golgi transport), which functionally interact with Ypt3.

View larger version (45K): [in this window] [in a new window]   Figure 4  Acid phosphatase glycosylation and carboxypeptidase Y sorting. (A) Effects of the over-expression of the dominant-negative Ryh1T25N mutant on acid phosphatase glycosylation in wild-type and ypt3-i5 mutant cells. The dominant-negative mutants were expressed in wild-type (wt) and ypt3-i5 mutant cells, and cells exponentially growing in EMM medium with 4 µM thiamine were transferred to EMM liquid medium without thiamine. Cells were incubated for 20 h at 27 °C before the acid phosphatase induction, and harvested. Then, acid phosphatase staining was performed. (B) The dominant-negative rab mutants, Ryh1T25N and Ypt3S24N (Cheng et al. 2002) were expressed in wild-type (wt), ryh1-i6 and ypt3-i5 mutant cells, and cells exponentially growing in EMM medium with 4 µM thiamine were transferred to EMM plate without thiamine. Cells were incubated for 20 h at 27 °C before immunoblot analysis. (C) Processing of carboxypeptidase Y in vivo. Wild-type strain and ryh1-i6 mutant cells were pulse-labeled with Express-35S-label for 10 min at 28 °C and chased. The immunoprecipitates were separated on an SDS-10% polyacrylamide gel. The autoradiograms of the fixed dried gels are shown.

The ryh1-i6 mutant shows defect in the secretion of acid phosphatase

As described above, ryh1-i6 mutant showed impaired glycosylation of acid phosphatase, and GFP-Ryh1Q70L was shown to localize at the plasma membrane. These prompted us to examine the transport of acid phosphatase, a protein that transits through the secretory pathway in the ryh1-i6 and ypt3-i5 cells. As shown in Fig. 5, both ryh1-i6 and ypt3-i5 mutants exhibited defects in acid phosphatase secretion. Interestingly, ryh1-i6 mutant showed a marked decrease in acid phosphatase secretion compared with that of ypt3-i5 mutant. The ryh1-i6 mutant cells secreted much less acid phosphatase than the wild-type or ypt3-i5 cells even at the permissive temperature of 27 °C (Fig. 5A). The expression of Ryh1Q70L (+ pryh1Q70L) in the ryh1-i6 mutant increased the acid phosphatase secretion to the same level as that of the wild-type Ryh1 (+ pryh1⁺) at 27 °C (Fig. 5B). Over-expression of ykt6⁺ (+ pykt6⁺) partially, but significantly, increased the acid phosphatase secretion (Fig. 5B). On the other hand, over-expression of ykt6⁺ had no effect on the defective acid phosphatase secretion of the ypt3-i5 cells (Fig. 5C). These results suggest that Ryh1 and Ypt3 are both involved in the acid phosphatase secretion from Golgi to the plasma membrane.

View larger version (19K): [in this window] [in a new window]   Figure 5  The ryh1-i6 mutant cells show defects in acid phosphatase secretion. (A) Wild-type, ypt3-i5 mutant, ryh1-i6 mutant, and ryh1 cells were assayed for secreted acid phosphatase activity as indicated. The data represent mean ± standard deviation of 10 determinations. (B) Over-expression of ykt6+ gene or Ryh1Q70L suppressed the acid phosphatase secretion defects of ryh1-i6 mutant cells. Cells were assayed for secreted acid phosphatase activity as indicated at 27 °C. The data represent mean ± standard deviation of 10 determinations. (C) Over-expression of ykt6+ gene had no effect on the acid phosphatase secretion defects of ypt3-i5 mutant cells. Cells were assayed for secreted acid phosphatase activity as indicated at 33 °C. The data represent mean ± standard deviation of 10 determinations.

A study by Aiba et al. (1998) reported the isolation of a high-osmolarity sensitive mutant allele of ryh1⁺. This prompted us to examine the effects of high osmolarity on the ryh1-i6 mutant. As shown in Fig. 6A, the addition of 1.2 M sorbitol inhibited the growth of the ryh1-i6 mutant cells at 27 °C. Unexpectedly, upon temperature upshift to 36 °C, sorbitol suppressed the temperature-sensitive growth defect of the ryh1-i6 mutant cells, suggesting a defect in cell wall integrity. The ryh1 cells showed almost the same phenotypes as those of the ryh1-i6 mutant cells (data not shown).

View larger version (44K): [in this window] [in a new window]   Figure 6  The ryh1-i6 mutant cells are defective in cell wall integrity. (A) Rescue of the ryh1-i6 mutant phenotype with an osmotic stabilizer. Wild-type and ryh1-i6 mutant cells were spotted onto YPD plate with or without 1.2 M sorbitol, and were incubated as described in Figure 2. (B) Cell wall digestion of wild-type, ypt3-i5 mutant and ryh1-i6 mutant cells by ß-glucanase. Cells exponentially growing in YPD medium were harvested and incubated with ß-glucanase (Zymolyase) at 30 °C with vigorous shaking. Cell lysis was monitored by the measurement of optical density at 660 nm (the value before adding the enzyme was taken as 100%). (C, D) The ypt3-i5 and ryh1-i6(C) mutants showed hypersensitivity to micafungin, a (1,3)-beta-D-glucan synthase inhibitor. Over-expression of the dominant-negative Ryh1T25N enhanced the hypersensitive phenotype of the ypt3-i5 mutant (D). Cells were spotted onto YPD, YPD plus 0.4 or 0.9 µg/mL micafungin and incubated at 27 °C for 4 days.

In this light, we then examined the effect of micafungin, an inhibitor of (1,3)-beta-D-glucan synthase (Carver 2004; Deng et al. 2005), on the ryh1-i6 and ypt3-i5 mutants (Fig. 6C,D). At low concentration of micafungin (0.4 µg/mL), the growth of ypt3-i5 mutant cells were markedly inhibited as compared with those of the ryh1-i6 mutant cells which were inhibited at higher concentration (0.9 µg/mL), in agreement with the above ß-glucanase experiment. Notably, the attenuated over-expression of the dominant-negative mutant protein Ryh1T25N in ypt3-i5 mutant cells in the presence of thiamine further enhanced the micafungin sensitivity of the ypt3-i5 mutant (Fig. 6D).

The ryh1-i6 mutant shows defects in the recycling from endosome to the Golgi and in trafficking from the Golgi to the plasma membrane

To investigate the role of Ryh1 in membrane trafficking, we visualized Syb1, the synaptobrevin of fission yeast, as a GFP-fusion protein (Edamatsu & Toyoshima 2003; Kita et al. 2004). In wild-type cells, GFP-Syb1 was visibly seen on the plasma membrane with more prominence at the growing ends of the cells (arrows, Fig. 7A), and was also brightly seen in the cytoplasm (Fig. 7A) wherein the observed dots fluoresced with the FM4-64 labeling dye that correspond to the Golgi/endosome compartments as previously described (Kita et al. 2004) (data not shown).

View larger version (57K): [in this window] [in a new window]   Figure 7  The ryh1-i6 mutant shows defects in multiple membrane trafficking steps. (A) GFP-fused synaptobrevin failed to localize at the cell surface and was degraded in the vacuole in the ryh1-i6 mutant cells. Wild-type (wt), ryh1-i6 mutant, isp6, double mutant of ryh1-i6 mutant and isp6 (ryh1-i6isp6) cells expressing GFP-Syb1 were cultured in YPD medium at 27 °C, and were examined by fluorescence microscopy. Arrows indicate GFP fluorescence associated with the plasma membrane. Bar, 10 µm. (B, C). Immunoblot analysis of GFP-Syb1. Wild-type (wt), ypt3-i5 mutant, ryh1-i6 mutant, isp6 cells, double mutant of ryh1-i6 mutant and isp6 cells (ryh1-i6isp6) expressing GFP-Syb1 were cultured in EMM containing 4 µM thiamine at 27 °C. The respective proteins were separated by SDS-PAGE and analyzed by immunoblotting using anti-GFP antiserum. Endogenous Cdc4 was used as a loading control and was immunoblotted using anti-Cdc4 antiserum. (D) Effect of latrunculin (Lat-A) treatment on GFP-Syb1 localization. The cells as indicated expressing GFP-Syb1 were processed as described in Figure 7 A and incubated with 100 µM Lat-A for 1 h at 27 °C, and were examined by fluorescence microscopy. Arrows indicate GFP fluorescence associated with the plasma membrane. Bar, 10 µm.

In agreement with the above hypothesis, when the isp6⁺ gene encoding a homolog of budding yeast vacuolar protease B was disrupted in the ryh1-i6 mutant cells, the level of GFP-Syb1 was restored to the level of GFP-Syb1 found in wild-type cells, while the endogenous level of Cdc4 in these cells remained constant (Fig. 7C). The isp6 cells exhibited stronger GFP-Syb1 fluorescence consistent with their higher level of GFP-Syb1 protein (Fig. 7 A,C). The isp6 cells were not defective in endocytosis (data not shown). Enhanced hazy or dotty pattern and membrane fluorescence were seen in ryh1-i6isp6 double mutant cells when observed microscopically (Fig. 7A, lower panels). Interestingly, this double mutant was more sensitive to high temperature, FK506 and Ca²⁺ than the single ryh1-i6 mutant cells (data not shown). These results suggest that the vacuolar protease is required to maintain the viability of the ryh1-i6 cells under stress condition, and that the deletion of the protease restores the apparent protein level of GFP-Syb1, but it exacerbates the defective recycling of the ryh1-i6 mutation. There were two bands evident in isp6 and ryh1-i6isp6 extracts for GFP-Syb1. The lower band, which was absent from the wild-type extracts (Fig. 7C), suggest that Isp6 is implicated in the physiological degradation of Syb1, and that the lower band may represent the extra-vacuolar degradation product of GFP-Syb1.

Membrane trafficking defect of ryh1-i6 mutant was further investigated using the actin-disrupting drug latrunculin-A (Lat-A). In wild-type cells, treatment with low levels of Lat-A, which predominantly blocks endocytosis (Valdivia et al. 2002), resulted in an accumulation of GFP-Syb1 at the plasma membrane (Fig. 7D). In contrast, in ypt3-i5 mutant cells, Lat-A treatment had no effect on the distribution of GFP-Syb1 in ypt3-i5 mutant and no accumulation of GFP-Syb1 was observed upon treatment (Fig. 7D), suggesting that the transport of GFP-Syb1 from the Golgi/endosome to the plasma membrane was mainly impaired. In ryh1-i6 mutant cells, accumulation of GFP-Syb1 at the plasma membrane was weakly observed by the Lat-A treatment (Fig. 7D, arrows), but intracellular GFP fluorescence was still brightly observed (Fig. 7D), suggesting that both the exit of the SNARE Syb1 from the Golgi and the recycling of Syb1 from endosome to the Golgi were impaired in this mutant.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
Rab proteins exist in all eukaryotic cells and form the largest branch of the small GTP-binding protein superfamily. The human genome contains 60 Rab proteins (Bock et al. 2001). The budding yeast S. cerevisiae genome sequence encodes 11 Rab proteins, and the fission yeast S. pombe genome sequence encodes only 7 Rab proteins including Ryh1.

In spite of the small number of Rab family protein in fission yeast, Ryh1 is not essential for cell viability (Aiba et al. 1998; Hengst et al. 1990). The finding that ryh1⁺ gene is not essential for cell viability may be due to its functional overlapping with other Rab proteins. Consistently, in the present study we showed the genetic interaction between ryh1⁺ and ypt3⁺, suggesting their functional overlapping. Interestingly, no genetic interaction was observed between their homologs in budding yeast. Luo & Gallwitz (2003) reported that temperature-sensitive ypt6-2 mutant showed no genetic interaction with ypt32A141D ypt31 double mutant.

In the present study, we showed that Ryh1 is implicated in retrograde traffic from endosome to the Golgi, similar to its budding yeast homolog Ypt6. Ryh1 may also be involved in the transport to the plasma membrane since the Ryh1Q70L localized not only at the Golgi/endosome, but also at the plasma membrane. Furthermore, the defects in cytokinesis (data not shown) and in cell wall integrity, and the decreased secretion of acid phosphatase were also observed in the ryh1-i6 mutant cells, and these may be reflective of the defects in the later transport step to the plasma membrane, a function possibly shared with Ypt3. Also, it was shown that Ryh1 is required for the exit of SNARE Syb1 from the Golgi as well as for the recycling of Syb1 from the endosome to the Golgi. Together, these results suggest that Ryh1 is involved in trafficking from Golgi/endosome to the plasma membrane in addition to its role in recycling.

A recent study by Chen et al. (2005) showed that Ypt31 and Ypt32, budding yeast homologs of Ypt3, regulate the recycling of Snc1, a budding yeast homolog of Syb1. However, in our present study, in contrast to the marked decrease of GFP-Syb1 level in ryh1-i6 mutant cells, the level of GFP-Syb1 in ypt3-i5 mutant was similar to that of the wild-type cells. In addition, treatment of ypt3-i5 mutant cells with low levels of Lat-A, which predominantly blocks endocytosis (Valdivia et al. 2002), did not affect the localization of GFP-Syb1 in ypt3-i5 mutant. These results suggest that, unlike budding yeast Ypt31 or Ypt32, Ypt3 does not regulate the recycling of Syb1.

In the present study, we show that the over-expression of Ryh1T25N, the GDP conformation mutant, inhibited the growth of ypt3-i5 mutant, and that it impaired acid phosphatase glycosylation of the ypt3-i5 mutant. We also show that both GFP-Ryh1 and GFP-Ypt3 localized at the Golgi/endosome. These results suggest that Ryh1 and Ypt3 are cooperatively involved in the protein glycosylation by the regulation of membrane trafficking events in the Golgi/endosome. Furthermore, the GFP-Ryh1 localization in ypt3-i5 mutant and the GFP-Ypt3 localization in ryh1-i6 mutant were both distinct from those in the wild-type cells. These results suggest that the other Rab protein regulates the proper localization of a specific Rab protein.

The possibility, however, that the two proteins function in parallel interacting trafficking pathways cannot be excluded in accounting for the genetic interaction. The impairment of Golgi/endosomal cycling caused by the mutation of ryh1⁺ gene may exacerbate the defects in secretion caused by the mutation of ypt3⁺ gene and vice versa. The convergence of these pathways and the importance for cell wall synthesis may explain the fact that alleles of each gene were isolated as FK506 hypersensitive mutants since FK506 inhibits calcineurin.

Interestingly, the multicopy suppressor, ykt6⁺, and the GTPase-deficient Ryh1 (Ryh1Q70L) suppressed the different phenotypes of the ryh1-i6 mutant cells. Budding yeast Ykt6 is implicated in multiple transport steps in the secretory pathway (Tsui & Banfield 2000; Dilcher et al. 2001; Lewis & Pelham 2002; Kweon et al. 2003; McNew et al. 1997). We hypothesized that fission yeast Ykt6 shares an overlapping function with Ryh1 in one or several cellular compartments where their function is associated with the temperature-sensitive phenotype of the ryh1-i6 mutant.

As described above, Ryh1Q70L reversed the impaired acid phosphatase secretion and the aberrant GFP-Syb1 localization (data not shown), and it also suppressed the FK506 and Ca²⁺ sensitivities of the ryh1-i6 mutant. These results suggest that the Ryh1Q70L mutant is physiologically efficient in the cellular processes related to these phenotypes. On the other hand, Ryh1Q70L failed to suppress the temperature sensitivity of the ryh1-i6 mutant. It is suggested that Ryh1 needs to be considerably inactivated for its role in the cellular compartment related to temperature sensitivity.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Strains, media and genetic and molecular biology methods

Schizosaccharomyces pombe strains used in this study are listed in Table 1. The complete medium, yeast extract-peptone-dextrose (YPD) and the minimal medium, Edinburgh minimum medium (EMM), have been previously described (Toda et al. 1996; Moreno et al. 1991). Standard genetic and recombinant-DNA methods (Moreno et al. 1991) were used except where noted. FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). The its6-1/ryh1-i6 mutant was isolated in a screen of cells that had been mutagenized with nitrosoguanidine as previously described (Zhang et al. 2000).

Database searches were performed using the National Center for Biotechnology Information BLAST network service (http://www.ncbi.nlm.nih.gov) and the Sanger Center S. pombe database search service (http://www.sanger.ac.uk).

Gene cloning

To clone the its6⁺ gene, the its6-1 mutant (KP1447) was grown at 27 °C and transformed with an S. pombe genomic DNA library constructed in the pDB248 vector. Leu⁺ transformants were replica-plated on to YPD plates at 36 °C and the plasmid DNA was recovered from transformants that showed plasmid-dependent rescue. By DNA sequencing, the suppressing plasmids fell into two classes, with one class containing the ryh1⁺ gene (SPAC4C5.02c) which encodes a protein that is highly similar to the mammalian Rab6 and to the budding yeast Ypt6, and the other class containing ykt6⁺ gene (SPBC13G1.11) which encodes a homolog of a budding yeast SNARE implicated in multiple transport steps in the secretory pathway.

To investigate the relationship between the cloned genes and its6-1 mutant, linkage analysis was performed as follows. The genes were subcloned into the pUC-derived plasmid containing Saccharomyces cerevisiaeLEU2 gene and integrated by homologous recombination into the genome of the wild-type strain HM123. The integrants were mated with the its6-1 mutant. The resulting diploid was sporulated, and the tetrads were dissected. When LEU2 gene was integrated into the ryh1⁺ gene allele of HM123 and mated with the its6-1 mutant, only parental ditype tetrads were found, indicating the allelism between the ryh1⁺ gene and the its6-1 mutation (data not shown). The same experiments were performed with ykt6⁺ gene, and the results showed that the ykt6⁺ gene is a multicopy suppressor of its6-1/ryh1-i6 mutant. The ryh1⁺ gene was disrupted as previously described (Hengst et al. 1990; Aiba et al. 1998).

Gene expression

For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (Maundrell 1993). Expression was repressed by the addition of 4 µg/mL thiamine to EMM, and was induced by washing and incubating the cells in EMM lacking thiamine. Genes either tagged or non-tagged were subcloned into pREP1, or pREP41 vectors to express the gene at various levels. Maximum expression of the fused gene was obtained using pREP1, while pREP41 contained the attenuated version of the nmt1 promoter (Basi et al. 1993).

To express green fluorescent protein (GFP)-Ryh1 or glutathione-S-transferase (GST)-Ryh1, the complete open reading frame of ryh1⁺ was amplified by PCR and was ligated to the C-terminus of the GFP carrying the S65T mutation (Heim et al. 1995) or to the C-terminus of the GST. To examine the functional activity of GFP-Ryh1 or GST-Ryh1, the fused gene was subcloned into the pREP1 expression vector. To obtain the chromosome-borne GFP-Ryh1, the fused gene was subcloned into the vector containing the ura4⁺ marker under the control of nmt1 promoter and was integrated into the chromosome at the ura4⁺ gene locus of KP1248 (h⁺leu1-32 ura4-294) as described (Cheng et al. 2002; Kita et al. 2004). GST-Ypt3 was similarly constructed by ligating the open reading frame of ypt3⁺ to the C-terminus of the GST. GFP-Ypt3 and GFP-Syb1 were expressed as described (Cheng et al. 2002; Kita et al. 2004). Krp1-RFP was similarly constructed by ligating the open reading frame of krp1⁺ encoding a furin homolog that resides in the Golgi (Powner & Davey 1998) to the N-terminus of the monomeric RFP (GENBANK Accession No. AB166761), which was designed based on the amino acid sequence of monomeric RFP (Accession No. AF506027) with an optimum codon usage for tobacco and Arabidopsis and no six-base cutting restriction sites except EcoRV. When the cells were cultured for microscopic observation, thiamine was added to the media to minimize the gene expression and to rule out any artifact by over-expression.

Microscopy

Methods in light microscopy, such as fluorescence microscopy and differential interference contrast microscopy which were used to observe the localization of GFP-tagged proteins and FM4-64 labeling, were performed as described (Cheng et al. 2002; Kita et al. 2004).

Site-directed mutagenesis

Ryh1T25N and Ryh1Q70L were generated using the Quick Change mutagenesis kit (Stratagene, La Jolla, CA, USA). In the amplification reaction, the mutant primers 5'-GGT GAG CAG TCA GTT GGC AAG AAT TCG TTA ATT ACA CGA TTT ATG-3' and 5'-CAT AAA TCG TGT AAT TAA CGA ATT CTT GCC AAC TGA CTG CTC ACC-3' were used to change Thr25 (ACA) into Gln (AAT) for Ryh1T25N. Also, the mutant primers 5'-CTT CAA CTA TGG GAT ACT GCT GGT CTC GAG CGT TTC CGT TCT TTA ATT C-3' and 5'-GAA TTA AAG AAC GGA AAC GCT CGA GAC CAG CAG TAT CCC ATC GTT GAA G-3' were used to change Gln70 (CAA) into Leu (CTC) for Ryh1Q70L. The chromosome-borne GFP-Ryh1Q70L was prepared as described above.

Assays and miscellaneous methods

Tetrad analysis (Zhang et al. 2000), acid phosphatase secretion assay (Kita et al. 2004), Western analysis (Ogiso et al. 2004), acid phosphatase staining (Maeda et al. 2004), pulse-chase analysis (Cheng et al. 2002), and immunoblot analysis of the S. pombe Cpy1 protein (Cheng et al. 2002; Maeda et al. 2004) were performed as previously described. Antibody to the fission yeast Cdc4 protein was prepared by immunizing the rabbit with purified Cdc4 protein, and was used for the detection of endogenous Cdc4 as a loading control.

	Acknowledgements

 
We thank Takashi Toda, Mitsuhiro Yanagida, and Chikashi Shimoda for providing strains and plasmids, and Susie O. Sio for critical reading of the manuscript, and Fujisawa JAPAN Inc. for gifts of FK506. This work was supported by 21st Century COE Program and research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Yoshimi Takai

^* Correspondence: E-mail: tkuno{at}med.kobe-u.ac.jp

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Received: 16 November 2005
Accepted: 27 November 2005

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