RESEARCH ARTICLE
Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe Ken-ichi Yoritsune, Yujiro Higuchi, Tomohiko Matsuzawa & Kaoru Takegawa Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Correspondence: Kaoru Takegawa, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Tel./fax: +81 92 642 2851; e-mail: [email protected] Present address: Tomohiko Matsuzawa, Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Received 23 July 2014; revised 26 August 2014; accepted 29 August 2014. Final version published online 19 September 2014. DOI: 10.1111/1567-1364.12207 Editor: Guenther Daum
YEAST RESEARCH
Keywords Golgi apparatus; N-glycan; oligosaccharide; phosphoenolpyruvate; Schizosaccharomyces pombe; transporter.
Abstract The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell–cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1+, pet2+, and pet3+) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1+ (pet1D) or in pet2+ (pet2D), but not the strain carrying a disruption in pet3+ (pet3D), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1D phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1D phenotype. Furthermore, the reduction in cell surface negative charge in pet1D cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi.
Introduction The addition of oligosaccharides to proteins is one of the major posttranslational modifications that take place in the endoplasmic reticulum (ER) and Golgi apparatus. Generally, this protein modification and subsequent processing of oligosaccharides, such as N-linked glycosylation, adds several important features to the character and/or function of the protein: increase in hydrophilicity, protection from proteases, proper targeting to the destination, and adjustment for interactors. Glycoproteins are also known to be involved in cellular recognition, agglutination, and infection. Interestingly, a large number of structurally different oligosaccharides have been found in living organisms, and these structurally different oligosaccharides are thought to have different functions.
FEMS Yeast Res 14 (2014) 1101–1109
There is a notable difference even between the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae so far as the oligosaccharides are concerned, which is the existence of pyruvylated galactose (PvGal) in S. pombe (Gemmill & Trimble, 1996, 1998). The cell surface of S. pombe is known to be negatively charged because of the presence of PvGal on the cell surface. In other yeast species, including S. cerevisiae, mannosylphosphate contributes to making the cell surface negatively charged (Parolis et al., 1996; Odani et al., 1997; Gemmill & Trimble, 1999). The negative charge of the cell surface is thought to be crucial for the fluidity and interaction of cells – for example, for flocculation and pathogenic adherence (Spillmann et al., 1993; Tanaka & Takegawa, 2001; Matsuzawa et al., 2011). We previously demonstrated that pyruvyltransferase Pvg1p could add pyruvate onto Gal residues of oligosaccharides, and this process was thought to occur in the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
1102
lumen of the Golgi apparatus, even though actual localization of Pvg1p was not known at that time (Yoritsune et al., 2013). The substrate for Pvg1p is phosphoenolpyruvate (PEP), which is synthesized during glycolysis and exists in the cytoplasm. Therefore, PEP ought to be transported into the lumen of the Golgi apparatus to be utilized as the substrate for Pvg1p. For this purpose, there must be transporter(s) that could bring PEP into the Golgi apparatus. In Arabidopsis thaliana, there are two PEP transporters, AtPPT1 and AtPPT2 (PEP/phosphate translocator), both of which were shown to be located in the plastidic inner envelope membrane (Fischer et al., 1997; Knappe et al., 2003). However, there is no report available so far describing the presence of any PEP transporter(s) in the Golgi apparatus of S. pombe. In this study, we searched for S. pombe homologs of the well-characterized A. thaliana PEP transporter AtPPT1. Consequently, we found three potential PEP transporter-encoding genes in the S. pombe genome, and named them as pet1+, pet2+, and pet3+. We found that the pet1D and pet2D cells, but not the pet3D cells, contained less amount of pyruvate on their cell surface, just like the pvg1D cells, suggesting that Pet1p and Pet2p, but not Pet3p, may have roles in transporting PEP into the Golgi apparatus. Consequently, we showed that expression of green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants restored the pyruvate level on the cell surface. We also confirmed that both Pet1p and Pet2p were localized to the Golgi apparatus. Most importantly, expression of either AtPPT1 or AtPPT2 in S. pombe and external addition of recombinant Pvg1p to the living cells suppressed the phenotype of pet1D cells. Taken together, our results suggested that Pet1p and Pet2p may function as primary and secondary PEP transporters, respectively, on the Golgi membrane of S. pombe.
Materials and methods Generation of strains and plasmids
Escherichia coli strain XL-1 Blue (Stratagene) was used for all general recombinant DNA cloning procedures. Schizosaccharomyces pombe strains used in this study are summarized in Table 1. The strain ARC039 (h leu1-32 ura4-C190T) was provided by Yuko Giga-Hama (Asahi Glass, Japan) and used as the wild type. Growth media and general experimental procedures for S. pombe were same as described previously (Alfa et al., 1993; Yoritsune et al., 2013). To delete the endogenous pet1+, pet2+, and pet3+ genes in the wild-type S. pombe strain ARC039, each gene was substituted by the ura4+ gene. To achieve this goal, pet1+, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
pet2+, and pet3+ genes were individually amplified from the S. pombe genomic DNA by PCR using the following oligonucleotides pairs: 50 -CGTATGTGCTTTTCTTGAAG CTGTCACC-30 and 50 -CATACCGGTGATAATTAAACG AACCAAC-30 for pet1+; 50 -GACCGCTAAGAATACTAGT TATCCTATTA-30 and 50 -GTCCAACTATACACAATGC TAAATTTG-30 for pet2+; 50 -TCCACCAATTGTGAACAAGAAAGCAAG-30 and 50 -GCCAATGAATAAGGATAGAAGACATTCG-30 for pet3+. Each of these PCR amplified products was separately ligated to the Promega pGEM-T vector. Next, we deleted portions of the pet1+, pet2+, and pet3+ open reading frames (ORFs) by digesting the respective plasmid DNA with XhoI/ClaI (for pet1+), EcoRI/KpnI (for pet2+), and HindIII (for pet3+) and then inserted a ura4+ DNA cassette into each linearized plasmid construct. Resultant plasmids, each carrying one of the deleted pet+ genes (pet1+, pet2+ or pet3+) as well as the ura4+ gene, were linearized, a portion of each linearized plasmid DNA was used for transforming the S. pombe ARC039 strain and subsequently ura4+ transformants were selected. The ura4+ transformants were further assessed by PCR to confirm that proper integration has indeed disrupted the desired pet+ gene. Plasmids pJK148-GFP-Pvg1p and pJK148-Pvg1p-GFP were constructed as follows. The pvg1+ gene, containing the pvg1+ ORF and its native promoter (1.5 kb) and terminator (0.5 kb) region, was amplified using the following pair of PCR primers: 50 -GTTTTGTCGACTTAAAAGA TGACGGGGCG-30 and 50 -GTTTTCCCGGGGTTGAGAA CCCTATTCCAC-30 . The PCR product, which contained a SalI and a BamHI restriction enzyme sites at the two ends of the DNA fragment, was digested with these two restriction enzymes, and the digested fragment was cloned into the corresponding sites of the plasmid pJK148. Subsequently, the gfp ORF, with or without stop codon, was fused to the 3’-end or 5’-end of the pvg1+ ORF of the resulting plasmid to create the expression plasmids pJK148-Pvg1p-GFP and pJK148-GFP-Pgv1p, respectively. Following a similar strategy and using the following two PCR primers, we created two other plasmids pJK148GFP-Pet1p and pJK148-Pet1p-GFP: 50 -GTTTTGAATTC GCTTTCTTGTCCGGTTTCACACACC-30 and 50 -GTTTT GGATCCCAGCATATCAAAGCAGCATACAC-30 . In this case, the PCR product contained an EcoRI and a BamHI restriction sites at the two ends of the DNA fragment, which was digested with these two restriction enzymes and the digested fragment was cloned into the corresponding restriction sites of pJK148 to create the plasmid pJK148-Pet1p. Thereafter, the gfp ORF was introduced appropriately as before to create the plasmids pJK148GFP-Pet1p and pJK148-Pet1p-GFP. In a similar manner, plasmids pJK148-GFP-Pet2p and pJK148-Pet2p-GFP were constructed using the following two PCR primers: FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1103
Table 1. Strains used in this study Strain
Genotype
ARC039 pvg1D pvg1D_GPvg1 pvg1D_Pvg1G GPvg1_Gms1R pet1D pet2D pet3D pet1D_pet2D pet1D_GPet1 pet1D_Pet1G pet2D_GPet2 pet2D_Pet2G Pet1G_Gms1R pet1D_GPPT1 pet1D_GPPT2 pet1D_GcPPT1 pet1D_GcPPT2 pet1D_cPPT1 pet1D_cPPT2 pet1D_NPet1
h h h h h h h h h h h h h h h h h h h h h
leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32
Sources ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T
pvg1::ura4+ pvg1::ura4+ gfp-pvg1-leu1+ pvg1::ura4+ pvg1-gfp-leu1+ pvg1D gfp-pvg1-leu1+ gms1-rfp-ura4+ pet1::ura4+ pet2::ura4+ pet3::ura4+ pet1D pet2::ura4+ pet1::ura4+ gfp-pet1-leu1+ pet1::ura4+ pet1-gfp-leu1+ pet2::ura4+ gfp-pet2-leu1+ pet2::ura4+ pet2-gfp-leu1+ pet1D pet1-gfp-leu1+ gms1-rfp-ura4+ pet1::ura4+ gfp-AtPPT1-leu1+ pet1::ura4+ gfp-AtPPT2-leu1+ pet1::ura4+ gfp-pet1(60aa)-AtPPT1(-90aa)-leu1+ pet1::ura4+ gfp-pet1(60aa)-AtPPT2(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-AtPPT1(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-AtPPT2(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-leu1+
50 -GTTTTCTGCAGCTGTTCTTGTTTATTAAATTGCGC-30 and 50 -GTTTTTTGCGGCCGCCATGTAATGCGAAAAGA AAGG-30 . To create the plasmids pJK148-AtPPT1 and pJK148-AtPPT2, the AtPPT1 and AtPPT2 ORFs were amplified using the following two sets of PCR primers: 50 -ATGCAAAGCTCCGCCGTATTCTCCCTCTCTC-30 and 50 -TTAAGCAGTCTTTGGCTTTGGCTTAATACC-30 for AtP PT1; 50 -ATGTTCGCTCTCACATTTCTAAATCCAAATC-30 and 50 -TCAAGACATTTTTGGATTTGGTTTGACTTG-30 for AtPPT2. The amplicons were used for replacing the pet1+ ORF in pJK148-Pet1p in such a manner that left the N-terminal 60 aa of Pet1p intact, resulting in plasmids pJK148-Pet1p60aa-AtPPT1 and pJK148-Pet1p60aa-AtPPT2. In addition, we created a plasmid construct pJK148-Pet1p60aa. Furthermore, the gfp ORF was introduced between the pet1+ promoter and start codon of AtPPTs and Pet1p in pJK148-AtPPT1, pJK148-AtPPT2, pJK148-Pet1p60aa-AtPPT1, and pJK148-Pet1p60aa-AtPPT2 to create the plasmids pJK148-GFP-AtPPT1, pJK 148-GFP-AtPPT2, pJK148-GFP-Pet1p60aa-AtPPT1, and pJK148-GFP-Pet1p60aa-AtPPT2, respectively. The plasmid pAU-Gms1p-RFP was prepared as described previously (Nakase et al., 2010). Fluorescence microscopy
To localize GFP- or RFP-tagged proteins, S. pombe cells expressing these proteins were grown overnight, cells were observed with a Nikon ECLIPSE 80i microscope (Nikon, Japan) and images of cells were captured with a Cool
FEMS Yeast Res 14 (2014) 1101–1109
Asahi Glass, Japan Matsuzawa et al. (2012) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study
SNAP CCD camera using the software METAMORPH (Roper Scientific, San Diego, CA). Alcian blue staining
To analyze negatively-charged glycan on the cell wall of S. pombe, procedures of Alcian blue staining and its data quantitation were performed as described previously with some modifications (Matsuzawa et al., 2012; Yoritsune et al., 2013). To add pyruvate onto Gal residues of oligosaccharides of cell wall, cells (OD600 of 2.0) were collected and washed three-times with water. Thereafter, cells were suspended in 500 lL of reaction solution [100 mM MOPS-NaOH (pH 6.0), 20 mM PEP monopotassium salt and 200 lg of recombinant Pvg1p] and incubated at 30 °C for 1 h, and subsequently, cells were stained using Alcian blue. Recombinant Pvg1p was prepared as described previously (Yoritsune et al., 2013).
Results Pvg1p localizes to the Golgi apparatus
To determine the cellular localization of Pvg1p, we added GFP tag to both the N- and C-termini of Pvg1p and expressed them in the pvg1D strain. We first checked the functionality of these GFP fusion proteins by staining the cells with Alcian blue, which binds to PvGal of S. pombe, and then determined the amount of PvGal on the cell surface (Matsuzawa et al., 2012). We found that the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
1104
Alcian blue stain level (μg per OD600 nm)
(a)
20 18 16 14 12 10 8 6 4 2 0
WT
pvg1Δ
pvg1Δ GFPPvg1p
pvg1Δ Pvg1pGFP
(b) Nomarski
GFP-Pvg1p
Gms1p-RFP
Merge
Fig. 1. Pvg1p localizes to the Golgi apparatus. (a) Alcian blue staining was carried out to determine the amount of PvGal on the cell surface of each indicated strain. The strain indicated here as pvg1D was used as a negative control. Three independent experiments were performed and results shown are mean standard error. (b) Cells expressing both GFP-Pvg1p and Gms1p-RFP were cultured overnight and observed using a fluorescent microscope. Gms1p was used as a marker protein for Golgi. In the merged image, GFP-Pvg1p colocalized with Gms1p-RFP, suggesting that Pvg1p is localized to the Golgi apparatus.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Construction of disruptants of putative PEP transporter genes
As the pyruvyltransferase Pvg1p is localized to the Golgi apparatus, PEP, which is the substrate of the enzyme, should also exist in the organelle. To explore the possibility that a PEP transporter is involved in transporting PEP from the cytoplasm to the Golgi apparatus, we exploited information available on the well-characterized AtPPT1 gene that encoded a PEP transporter in A. thaliana (Fischer et al., 1997; Knappe et al., 2003). By searching the S. pombe genome database (PomBase; http://www. pombase.org/), we identified following three genes that were homologous to AtPPT1: namely, SPAC22F8.04, SPBC83.11, and SPAC22E12.01, and designated them as pet1+, pet2+, and pet3+ (PEP transporter), respectively. The deduced amino acid sequences of Pet1p, Pet2p, and Pet3p exhibited 16%, 17%, and 20% sequence homology to AtPPT1, as determined by analyzing these sequences using CLUSTALW (ver2.1; http://clustalw.ddbj.nig.ac.jp/; Supporting Information, Fig. S1). To examine the function of each gene, we next generated three single gene disruptants, each carrying a disruption in the pet1+, pet2+, or pet3+ gene, and performed alcian blue staining assay to determine the amount of PvGal on the cell surface of each strain. As shown in Fig. 2, cell surface levels of PvGal in pvg1D and pet1D were significantly lower than that in the control cells; on the other hand, cell surface level of PvGal in pet2D was approximately two-thirds of that in the control cells, and cell surface levels of PvGal in pet3D and control cells were same. These results suggested that Pet1p primarily and Pet2p partially contribute to the production of PvGal on the cell surface, possibly by functioning as PEP transporters, whereas Pet3p has almost no role in this process. Therefore, we decided not
Alcian blue stain level (μg per OD600 nm)
parent strain pvg1D had significantly reduced level of PvGal on the cell surface than the control cells (Fig. 1a), as was reported earlier (Yoritsune et al., 2013). Although expression of GFP-Pvg1p (GFP fused to the N-terminal end of Pvg1p) in these cells fully restored the PvGal level on the cell surface, expression of Pvg1p-GFP (GFP fused to the C-terminal end of Pvg1p) failed to do so (Fig. 1a). Therefore, we examined the GFP-Pvg1p expressing cells by fluorescence microscopy to determine the cellular localization of Pvg1p and found that they were expressed as punctate structures (Fig. 1b). To verify whether Pvg1p is localized to the Golgi apparatus, we coexpressed GFPPvg1p and RFP-fused Gms1p, which is a UDP-Gal transporter and used as a Golgi marker protein (Tabuchi et al., 1997; Nakase et al., 2010). As shown in Fig. 1b, GFP-Pvg1p clearly colocalized with Gms1p-RFP. Taken together, these results suggested that Pvg1p is localized to the Golgi apparatus and is functionally active [i.e. adds pyruvate to the N-glycan oligosaccharides, as was demonstrated earlier by Yoritsune et al. (2013)].
20 18 16 14 12 10 8 6 4 2 0
WT
pvg1Δ
pet1Δ
pet2Δ
pet3Δ
pet1Δ pet2Δ
Fig. 2. Analysis of alcian blue stained petD strains. Alcian blue staining was carried out as described in Fig. 1a and the amount of PvGal on the cell surface of each strain was quantified. pvg1D, negative control. Results shown are mean standard error (n = 3).
FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1105
to use the pet3D strain for further analysis. We also constructed a pet1D/pet2D double disruptant strain, surface level of PvGal in which was further reduced than that in the single disruptant pet1D strain, suggesting that Pet1p and Pet2p might be working cooperatively.
and C-terminal GFP fusion constructs of Pet1p and Pet2p in the respective disruptants. All four fusion proteins showed the similar localization pattern (i.e. punctate structures), which indicated that they were probably localized to the Golgi apparatus (Fig. 3a). We also stained these cells with alcian blue and found that both GFP-Pet1p and Pet1p-GFP constructs complemented the phenotypic defect of the pet1D cells, and that the Pet2p-GFP fusion protein fully and GFPPet2p fusion protein partially complemented the phenotypic defect of the pet2D cells (Fig. 3b). Taken together, our results suggested that the GFP fusion constructs of Pet1p and Pet2p,
Both Pet1p and Pet2p localize to the Golgi apparatus
To determine the cellular localization of putative S. pombe PEP transporters Pet1p and Pet2p, we expressed N-terminal (a)
FEMS Yeast Res 14 (2014) 1101–1109
(b) Alcian blue stain level (μg per OD600 nm)
Fig. 3. GFP-fused Pet proteins are functional. (a) Schizosaccharomyces pombe pet1D cells expressing GFP-Pet1p or Pet1p-GFP and pet2D cells expressing GFP-Pet2p or Pet2p-GFP were cultured overnight and then observed using a fluorescent microscope. All four GFP fusion proteins appeared to be expressed as punctate structures. (b) Alcian blue staining of cells and the amount of PvGal on the cell surface of each strain was determined as described under Fig. 1a. Expression of Pet1p and Pet2p (as N- or C-terminal GFP fusion proteins) in S. pombe pet1D and pet2D cells, respectively, using plasmid constructs described in the Materials and methods, restored the PvGal level on the cell surface of each disruptant to that of the control cell level. Three independent experiments were carried out and results shown are mean standard error.
GFP-Pet1p
Nomarski
20 18 16 14 12 10 8 6 4 2 0
Nomarski Pet1p-GFP
Nomarski
GFP-Pet2p
Nomarski
Pet2p-GFP
WT
pet1Δ
pet1Δ pet1Δ pet2Δ GFP- Pet1pPet1p GFP
pet2Δ GFPPet2p
pet2Δ Pet2pGFP
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
1106
generated in this study, are functional, apart from GFPPet2p. To further confirm the localization of Pet1p, we expressed Gms1p-RFP in the Pet1p-GFP expressing strain. As shown in Fig. 4, Pet1p-GFP colocalized with the Gms1p-RFP in dot-like structures. Taken together, these results suggested that Pet1p, which is situated on the membrane of the Golgi apparatus, transports PEP from the cytoplasm into the organelle. Chimeric constructs of Pet1p and AtPPT proteins are able to suppress the phenotype of pet1D cells
So far, we have demonstrated that Pet1p is mainly responsible for the transport of PEP into the Golgi apparatus of S. pombe. To further examine whether Pet1p might function similarly as AtPPT1 and AtPPT2, both of which are well-characterized PEP transporters found in A. thaliana, we individually expressed these two plant PEP transporters in pet1D cells. Resultant strains expressing either full-length AtPPT1 or full-length AtPPT2 could not suppress the phenotype of pet1D cells (data not shown). Indeed, expression of GFP-tagged AtPPT1 and AtPPT2 in S. pombe showed ER-like localization (GFP-AtPPT1, Fig. 5a), probably because of these fusion proteins were not targeted properly from the ER to the Golgi apparatus. Protein sequence analysis using the SOSUI program (http://harrier.nagaha-
Nomarski
Pet1p-GFP
Merge
Gms1p-RFP
Fig. 4. Pet1p localizes to the Golgi apparatus. Gms1p-RFP expressing plasmid construct was introduced into the cells expressing Pet1p-GFP. The resultant cells were cultured overnight and observed using a fluorescent microscope. In the merged image, Pet1p-GFP colocalized with Gms1p-RFP, indicating that Pet1p is localized to the Golgi apparatus.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ma-i-bio.ac.jp/sosui/) revealed that Pet1p, AtPPT1, and AtPPT2 are predicted to contain 10, 7, and 5 transmembrane domains, respectively (Fig. 5b). In addition, each protein is predicted to contain an N-terminal cytoplasmic region. We reasoned that the differences in the N-terminal regions of Pet1p and AtPPTs might be responsible for the mistargeting of AtPPTs in S. pombe. Therefore, to increase the possibility of functional expression of these plant transporters in S. pombe, we replaced the N-terminal portions of the AtPPT1 and AtPPT2 with the N-terminal cytoplasmic region (containing first 60 amino acid residues) of Pet1p (Fig. 5c). We also fused GFP to the N-terminal end of each one of these two chimeric transporters. When expressed in pet1D cells, both GFP-Pet1p60aa-AtPPT1 and GFP-Pet1a60aa-ATPPT2 were expressed as punctate structures (Fig. 5d). Expression of these chimeric proteins also partially suppressed the phenotype of pet1D cells (Fig. 5e). To exclude the possibility that the N-terminal region of Pet1p might be sufficient in complementing the pet1D phenotype, we separately expressed the first 60 aa region of Pet1p in pet1D cells. Our results clearly showed that the expression of only 60 aa of Pet1p could not complement the phenotypic defect in pet1D cells (Fig. 5e), suggesting that rest of the sequence of either AtPPT1 or AtPPT2 protein, but not 60 aa of Pet1p, is sufficient to form a functional protein that could suppress the pet1D phenotype (Fig. 5e). Taken together, these results suggested that Pet1p functions as PEP transporters, just like the plant PEP transporters. Addition of recombinant Pvg1p to living cells can suppress the pet1D phenotype
Previously, we demonstrated that addition of recombinant Pvg1p to living cells could complement the pvg1D phenotype, as judged by alcian blue staining, likely because the addition of Pvg1p from outside could transfer pyruvate onto the Gal residues on the cell wall (Yoritsune et al., 2013). To investigate whether pet1D cells would behave similarly as the pvg1D cells, we repeated the same experiment using the pet1D cells. As shown in Fig. 6, the alcian blue staining level of pet1D cells was significantly increased following incubation with extracellularly added recombinant Pvg1p and PEP, and the increase in staining level was similar for the pet1D and pvg1D cells. This result suggested that the amount of N-linked b-Gal residues, which were surface exposed and not modified by pyruvate, was about same in both pet1D and pvg1D cells.
Discussion To understand the molecular mechanism of N-glycan protein synthesis, it is essential to determine where and FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1107
Fig. 5. Expressions of chimeric AtPPT proteins suppress the phenotype of the pet1D strain. (a) GFP-tagged AtPPT1 was expressed in the pet1D cells, and cells expressing the GFP fusion protein were observed using a fluorescent microscope. The fusion protein was likely localized to ER-like structures. (b) Transmembrane domains of Pet1p, AtPPT1, and AtPPT2, as predicted by SOSUI program, are shown. (c) Schematic diagram depicting the strategy for constructing the AtPPT1 chimera, where the N-terminal portion of AtPPT1 was replaced with the N-terminal portion (first 60 amino acid residues) of Pet1p. (d) GFP-tagged Pet1p60aa-AtPPT1 and Pet1p60aa-AtPPT2 chimeric proteins were individually expressed in the pet1D cells, and cells expressing the GFP fusion protein were observed using a fluorescent microscope. Both chimeric proteins appeared to be expressed as Golgi-like punctate structures. (e) Alcian blue staining and quantitation of PvGal on the cell surface of each strain was carried out as described before. Note that the expression of AtPPT1 or AtPPT2 chimeric construct partially suppressed the phenotype of the pet1D cells. However, complementation of phenotype was not observed in cells expressing only the first 60 amino acid residues of Pet1p. Three independent experiments were performed and results shown are mean standard error.
20 18 16 14 12 10 8 6 4 2 0 WT
(b)
pet1Δ
pet1Δ chimeric AtPPT1
AtPPT1
Pet1p
(c)
Pet1p
(d) Nomarski
AtPPT1
GFP-chimeric AtPPT1
pet1Δ chimeric AtPPT2
pet1Δ Pet1p 60 aa
AtPPT2
80 aa
100 aa
how oligosaccharide-modifying enzymes are regulated. In fission yeast, negative charge conferred by PvGal moieties of oligosaccharides is a major determinant of physiological cell response (Gemmill & Trimble, 1996; Tanaka & Takegawa, 2001; Matsuzawa et al., 2011). We have recently characterized the pyruvyltransferase Pvg1p; however, at that time, we did not examine where the enzyme was localized (Yoritsune et al., 2013). Therefore, at the beginning of this study, we expressed a functionally active, N-terminally GFP-tagged Pvg1p in S. pombe and found using fluorescence microscopy that this fusion protein was localized to the Golgi apparatus. Previously, Pvg1p was shown to be localized in the periplasmic space (Andreishcheva et al., 2004). In that study, however, a C-terminally GFP-tagged Pvg1p was used and improper fusion might have lead to its mislocalization. Thus, from our observation, we concluded that Pvg1p is localized to
FEMS Yeast Res 14 (2014) 1101–1109
(e) Alcian blue stain level (μg per OD600 nm)
(a) GFP-AtPPT1
Nomarski
Chimeric AtPPT1
GFP-chimeric AtPPT2
the Golgi apparatus, where it attaches pyruvate to the Gal residues of oligosaccharides. As PEP, from which pyruvate is produced, should be supplied to the lumen of the Golgi apparatus, we speculated that there ought to be transporter(s) for delivering the cytoplasmic PEP into the organelle. Based on the results of BLAST search against the A. thaliana PEP transporter AtPPT1, we found three possible candidate genes in S. pombe. According to the S. pombe genome database, Pet1p and Pet2p were predicted to be triose phosphate transporters, whereas Pet3p showed highest homology to a probable sugar phosphate/phosphate translocator in A. thaliana (NP_172135.2) with an e-value of 7e53. Indeed, results described in this study showed that Pet3p had no role in the modification of Gal to PvGal on the cell surface. On the other hand, Pet1p and Pet2p may play primary and secondary roles in producing PvGal on ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
Alcian blue stain level (μg per OD600 nm)
1108
20 18 16 14 12 10 8 6 4 2 0
Strain
WT
WT
pvg1 Δ pvg1 Δ pet1 Δ
pet1 Δ
Pvg1p Fig. 6. Addition of recombinant Pvg1p suppresses the phenotype of the pet1D strain. Cultures of indicated strains were incubated with or without recombinant Pvg1p and PEP at 30 °C for 1 h, and cells were then stained with alcian blue and the amount of PvGal on the cell surface was determined as before. Note that the addition of recombinant Pvg1p and PEP to the living pet1D cells increased the amount of PvGal on the cell surface. The strain pvg1D was used here as a control. Three independent experiments were performed and results shown are mean standard error.
the cell surface, respectively. In S. cerevisiae, negative charge of the cell surface is provided by mannosylphosphate, unlike S. pombe, and thus, it is not necessary for PEP to be transported into the Golgi apparatus. Consistent with this, in budding yeast, there is no ortholog for Pet1p and one for Pet2p with relatively low homology (YJL193W; e-value, 6.6e22). In other fungi, we found that the filamentous fungus Trichoderma reesei has the Pet1p (XP_006969429.1; e-value, 8e47), Pet2p (XP_0069 68103.1; e-value, 5e90), and Pvg1p (XP_006969404.1; e-value, 3e45) orthologs. Therefore, it is intriguing to investigate whether pyruvylated oligosaccharides exist in T. reesei like S. pombe. Analyses of substrate specificities of AtPPT1 and AtPPT2 showed that AtPPT1 preferred 2-phosphoglycerate and PEP over 3-phosphoglycerate as a substrate and AtPPT2 preferred PEP over 2-phosphoglycerate and 3-phosphoglycerate as a substrate (Knappe et al., 2003). Hence, it is possible that Pet1p and Pet2p in S. pombe may have different substrate specificities. Based on results described in this study, we believe that Pet1p has more preference for PEP as a substrate than Pet2p. Moreover, as we could not find any phenotype for the pet3D cells, Pet3p might be transporting a different substrate, such as a sugar phosphate, as was predicted by the BLAST search. We undertook to functionally express GFP-AtPPT fusion proteins in S. pombe, but initially these fusion proteins did not reach the Golgi apparatus and instead they ended up in the ER-like structures, likely because of unsuccessful targeting. In plant, both AtPPTs are localized in the chloroplast (Knappe et al., 2003); thus, they
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
probably lacked the signal for localizing to the Golgi apparatus in S. pombe. To assist the AtPPTs to reach the Golgi apparatus in fission yeast, we next replaced the N-terminal cytoplasmic regions of AtPPT1 and AtPPT2 with the first 60 aa of Pet1p. These chimeric constructs successfully reached the Golgi apparatus, suggesting that the N-terminal part of Pet1p contained the signal for Golgi localization. Although recent protein sequence analysis softwares could better predict the subcellular localization of proteins, predicting the localization of transmembrane proteins is still difficult, especially for the ones that are localized to the Golgi apparatus (Mukai et al., 2011). Thus, more detailed analyses of Pet1p and Pet2p may provide appropriate information regarding the Golgi localization sequence. In summary, we examined three putative S. pombe PEP transporter genes, one of which (i.e. pet3+) probably does not have the function of PEP transport. We demonstrated that Pet1p is mainly responsible for transporting PEP from the cytoplasm into the Golgi apparatus of S. pombe, and Pet2p may have a supporting role in PEP transport. In conjunction with the enzymatic characterization of Pvg1p that was carried out earlier, we have found out how pyruvate is attached to Gal residues of oligosaccharides. Further analysis of PvGal, including the structural analysis of Pvg1p, will be pursued to gain new insights in the field of glycobiology that could find applications in developing novel pharmaceutical agents.
Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (K.T.). The authors declare that they have no conflict of interest.
References Alfa C, Fantes P, Hyams J, McLeod M & Warbrick E (1993) Experiments with Fission Yeast. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Andreishcheva EN, Kunkel JP, Gemmill TR & Trimble RB (2004) Five genes involved in biosynthesis of the pyruvylated Galb1,3-epitope in Schizosaccharomyces pombe N-linked glycans. J Biol Chem 279: 35644–35655. Fischer K, Kammerer B, Gutensohn M, Arbinger B, Weber A, H€ausler RE & Fl€ ugge UI (1997) A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/ phosphate antiporter. Plant Cell 9: 453–462. Gemmill TR & Trimble RB (1996) Schizosaccharomyces pombe produces novel pyruvate-containing N-linked oligosaccharides. J Biol Chem 271: 25945–25949.
FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
Gemmill TR & Trimble RB (1998) All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4,6-O-[(R)-(1-carboxyethylidine)]-Galb1,3Gala1. Glycobiology 8: 1087–1095. Gemmill TR & Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426: 227–237. Knappe S, L€ ottgert T, Schneider A, Voll L, Fl€ ugge UI & Fischer K (2003) Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis – AtPPT1 may be involved in the provision of signals for correct mesophyll development. Plant J 36: 411– 420. Matsuzawa T, Morita T, Tanaka N, Tohda H & Takegawa K (2011) Identification of a galactose-specific flocculin essential for non-sexual flocculation and filamentous growth in Schizosaccharomyces pombe. Mol Microbiol 82: 1531–1544. Matsuzawa T, Yoritsune K & Takegawa K (2012) MADS box transcription factor Mbx2/Pvg4 regulates invasive growth and flocculation by inducing gsf2+ expression in fission yeast. Eukaryot Cell 11: 151–158. Mukai Y, Yoshizawa M, Sasaki T, Ikeda M, Tomii K, Hirokawa T & Suwa M (2011) Discrimination of Golgi type II membrane proteins based on their hydropathy profiles and the amino acid propensities of their transmembrane regions. Biosci Biotechnol Biochem 75: 82–88. Nakase M, Tani M, Morita T, Kitamoto HK, Kashiwazaki J, Nakamura T, Hosomi A, Tanaka N & Takegawa K (2010) Mannosylinositol phosphorylceramide is a major sphingolipid component and is required for proper localization of plasma-membrane proteins in Schizosaccharomyces pombe. J Cell Sci 123: 1578–1587.
FEMS Yeast Res 14 (2014) 1101–1109
1109
Odani T, Shimma Y, Wang XH & Jigami Y (1997) Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett 420: 186–190. Parolis LA, Duus JO, Parolis H, Meldal M & Bock K (1996) The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the structure of the phosphomannan backbone. Carbohydr Res 293: 101–117. Spillmann D, Hard K, Thomas-Oates J, Vliegenthart JF, Misevic G, Burger MM & Finne J (1993) Characterization of a novel pyruvylated carbohydrate unit implicated in the cell aggregation of the marine sponge Microciona prolifera. J Biol Chem 268: 13378–13387. Tabuchi M, Tanaka N, Iwahara S & Takegawa K (1997) The Schizosaccharomyces pombe gms1+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem Biophys Res Commun 232: 121–125. Tanaka N & Takegawa K (2001) Biosynthetic pathway and physiological role of galactose-containing oligosaccharides in fission yeast Schizosaccharomyces pombe. Trends Glycosci Glycotechnol 13: 519–532. Yoritsune K, Matsuzawa T, Ohashi T & Takegawa T (2013) The fission yeast Pvg1p has galactose-specific pyruvyltransferase activity. FEBS Lett 587: 917–921.
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Amino acid sequence alignments of Pet1p, Pet2p, Pet3p, and AtPPT1.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe Ken-ichi Yoritsune, Yujiro Higuchi, Tomohiko Matsuzawa & Kaoru Takegawa Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Correspondence: Kaoru Takegawa, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Tel./fax: +81 92 642 2851; e-mail: [email protected] Present address: Tomohiko Matsuzawa, Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Received 23 July 2014; revised 26 August 2014; accepted 29 August 2014. Final version published online 19 September 2014. DOI: 10.1111/1567-1364.12207 Editor: Guenther Daum
YEAST RESEARCH
Keywords Golgi apparatus; N-glycan; oligosaccharide; phosphoenolpyruvate; Schizosaccharomyces pombe; transporter.
Abstract The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell–cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1+, pet2+, and pet3+) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1+ (pet1D) or in pet2+ (pet2D), but not the strain carrying a disruption in pet3+ (pet3D), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1D phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1D phenotype. Furthermore, the reduction in cell surface negative charge in pet1D cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi.
Introduction The addition of oligosaccharides to proteins is one of the major posttranslational modifications that take place in the endoplasmic reticulum (ER) and Golgi apparatus. Generally, this protein modification and subsequent processing of oligosaccharides, such as N-linked glycosylation, adds several important features to the character and/or function of the protein: increase in hydrophilicity, protection from proteases, proper targeting to the destination, and adjustment for interactors. Glycoproteins are also known to be involved in cellular recognition, agglutination, and infection. Interestingly, a large number of structurally different oligosaccharides have been found in living organisms, and these structurally different oligosaccharides are thought to have different functions.
FEMS Yeast Res 14 (2014) 1101–1109
There is a notable difference even between the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae so far as the oligosaccharides are concerned, which is the existence of pyruvylated galactose (PvGal) in S. pombe (Gemmill & Trimble, 1996, 1998). The cell surface of S. pombe is known to be negatively charged because of the presence of PvGal on the cell surface. In other yeast species, including S. cerevisiae, mannosylphosphate contributes to making the cell surface negatively charged (Parolis et al., 1996; Odani et al., 1997; Gemmill & Trimble, 1999). The negative charge of the cell surface is thought to be crucial for the fluidity and interaction of cells – for example, for flocculation and pathogenic adherence (Spillmann et al., 1993; Tanaka & Takegawa, 2001; Matsuzawa et al., 2011). We previously demonstrated that pyruvyltransferase Pvg1p could add pyruvate onto Gal residues of oligosaccharides, and this process was thought to occur in the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
1102
lumen of the Golgi apparatus, even though actual localization of Pvg1p was not known at that time (Yoritsune et al., 2013). The substrate for Pvg1p is phosphoenolpyruvate (PEP), which is synthesized during glycolysis and exists in the cytoplasm. Therefore, PEP ought to be transported into the lumen of the Golgi apparatus to be utilized as the substrate for Pvg1p. For this purpose, there must be transporter(s) that could bring PEP into the Golgi apparatus. In Arabidopsis thaliana, there are two PEP transporters, AtPPT1 and AtPPT2 (PEP/phosphate translocator), both of which were shown to be located in the plastidic inner envelope membrane (Fischer et al., 1997; Knappe et al., 2003). However, there is no report available so far describing the presence of any PEP transporter(s) in the Golgi apparatus of S. pombe. In this study, we searched for S. pombe homologs of the well-characterized A. thaliana PEP transporter AtPPT1. Consequently, we found three potential PEP transporter-encoding genes in the S. pombe genome, and named them as pet1+, pet2+, and pet3+. We found that the pet1D and pet2D cells, but not the pet3D cells, contained less amount of pyruvate on their cell surface, just like the pvg1D cells, suggesting that Pet1p and Pet2p, but not Pet3p, may have roles in transporting PEP into the Golgi apparatus. Consequently, we showed that expression of green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants restored the pyruvate level on the cell surface. We also confirmed that both Pet1p and Pet2p were localized to the Golgi apparatus. Most importantly, expression of either AtPPT1 or AtPPT2 in S. pombe and external addition of recombinant Pvg1p to the living cells suppressed the phenotype of pet1D cells. Taken together, our results suggested that Pet1p and Pet2p may function as primary and secondary PEP transporters, respectively, on the Golgi membrane of S. pombe.
Materials and methods Generation of strains and plasmids
Escherichia coli strain XL-1 Blue (Stratagene) was used for all general recombinant DNA cloning procedures. Schizosaccharomyces pombe strains used in this study are summarized in Table 1. The strain ARC039 (h leu1-32 ura4-C190T) was provided by Yuko Giga-Hama (Asahi Glass, Japan) and used as the wild type. Growth media and general experimental procedures for S. pombe were same as described previously (Alfa et al., 1993; Yoritsune et al., 2013). To delete the endogenous pet1+, pet2+, and pet3+ genes in the wild-type S. pombe strain ARC039, each gene was substituted by the ura4+ gene. To achieve this goal, pet1+, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
pet2+, and pet3+ genes were individually amplified from the S. pombe genomic DNA by PCR using the following oligonucleotides pairs: 50 -CGTATGTGCTTTTCTTGAAG CTGTCACC-30 and 50 -CATACCGGTGATAATTAAACG AACCAAC-30 for pet1+; 50 -GACCGCTAAGAATACTAGT TATCCTATTA-30 and 50 -GTCCAACTATACACAATGC TAAATTTG-30 for pet2+; 50 -TCCACCAATTGTGAACAAGAAAGCAAG-30 and 50 -GCCAATGAATAAGGATAGAAGACATTCG-30 for pet3+. Each of these PCR amplified products was separately ligated to the Promega pGEM-T vector. Next, we deleted portions of the pet1+, pet2+, and pet3+ open reading frames (ORFs) by digesting the respective plasmid DNA with XhoI/ClaI (for pet1+), EcoRI/KpnI (for pet2+), and HindIII (for pet3+) and then inserted a ura4+ DNA cassette into each linearized plasmid construct. Resultant plasmids, each carrying one of the deleted pet+ genes (pet1+, pet2+ or pet3+) as well as the ura4+ gene, were linearized, a portion of each linearized plasmid DNA was used for transforming the S. pombe ARC039 strain and subsequently ura4+ transformants were selected. The ura4+ transformants were further assessed by PCR to confirm that proper integration has indeed disrupted the desired pet+ gene. Plasmids pJK148-GFP-Pvg1p and pJK148-Pvg1p-GFP were constructed as follows. The pvg1+ gene, containing the pvg1+ ORF and its native promoter (1.5 kb) and terminator (0.5 kb) region, was amplified using the following pair of PCR primers: 50 -GTTTTGTCGACTTAAAAGA TGACGGGGCG-30 and 50 -GTTTTCCCGGGGTTGAGAA CCCTATTCCAC-30 . The PCR product, which contained a SalI and a BamHI restriction enzyme sites at the two ends of the DNA fragment, was digested with these two restriction enzymes, and the digested fragment was cloned into the corresponding sites of the plasmid pJK148. Subsequently, the gfp ORF, with or without stop codon, was fused to the 3’-end or 5’-end of the pvg1+ ORF of the resulting plasmid to create the expression plasmids pJK148-Pvg1p-GFP and pJK148-GFP-Pgv1p, respectively. Following a similar strategy and using the following two PCR primers, we created two other plasmids pJK148GFP-Pet1p and pJK148-Pet1p-GFP: 50 -GTTTTGAATTC GCTTTCTTGTCCGGTTTCACACACC-30 and 50 -GTTTT GGATCCCAGCATATCAAAGCAGCATACAC-30 . In this case, the PCR product contained an EcoRI and a BamHI restriction sites at the two ends of the DNA fragment, which was digested with these two restriction enzymes and the digested fragment was cloned into the corresponding restriction sites of pJK148 to create the plasmid pJK148-Pet1p. Thereafter, the gfp ORF was introduced appropriately as before to create the plasmids pJK148GFP-Pet1p and pJK148-Pet1p-GFP. In a similar manner, plasmids pJK148-GFP-Pet2p and pJK148-Pet2p-GFP were constructed using the following two PCR primers: FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1103
Table 1. Strains used in this study Strain
Genotype
ARC039 pvg1D pvg1D_GPvg1 pvg1D_Pvg1G GPvg1_Gms1R pet1D pet2D pet3D pet1D_pet2D pet1D_GPet1 pet1D_Pet1G pet2D_GPet2 pet2D_Pet2G Pet1G_Gms1R pet1D_GPPT1 pet1D_GPPT2 pet1D_GcPPT1 pet1D_GcPPT2 pet1D_cPPT1 pet1D_cPPT2 pet1D_NPet1
h h h h h h h h h h h h h h h h h h h h h
leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32 leu1-32
Sources ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T ura4-C190T
pvg1::ura4+ pvg1::ura4+ gfp-pvg1-leu1+ pvg1::ura4+ pvg1-gfp-leu1+ pvg1D gfp-pvg1-leu1+ gms1-rfp-ura4+ pet1::ura4+ pet2::ura4+ pet3::ura4+ pet1D pet2::ura4+ pet1::ura4+ gfp-pet1-leu1+ pet1::ura4+ pet1-gfp-leu1+ pet2::ura4+ gfp-pet2-leu1+ pet2::ura4+ pet2-gfp-leu1+ pet1D pet1-gfp-leu1+ gms1-rfp-ura4+ pet1::ura4+ gfp-AtPPT1-leu1+ pet1::ura4+ gfp-AtPPT2-leu1+ pet1::ura4+ gfp-pet1(60aa)-AtPPT1(-90aa)-leu1+ pet1::ura4+ gfp-pet1(60aa)-AtPPT2(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-AtPPT1(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-AtPPT2(-90aa)-leu1+ pet1::ura4+ pet1(60aa)-leu1+
50 -GTTTTCTGCAGCTGTTCTTGTTTATTAAATTGCGC-30 and 50 -GTTTTTTGCGGCCGCCATGTAATGCGAAAAGA AAGG-30 . To create the plasmids pJK148-AtPPT1 and pJK148-AtPPT2, the AtPPT1 and AtPPT2 ORFs were amplified using the following two sets of PCR primers: 50 -ATGCAAAGCTCCGCCGTATTCTCCCTCTCTC-30 and 50 -TTAAGCAGTCTTTGGCTTTGGCTTAATACC-30 for AtP PT1; 50 -ATGTTCGCTCTCACATTTCTAAATCCAAATC-30 and 50 -TCAAGACATTTTTGGATTTGGTTTGACTTG-30 for AtPPT2. The amplicons were used for replacing the pet1+ ORF in pJK148-Pet1p in such a manner that left the N-terminal 60 aa of Pet1p intact, resulting in plasmids pJK148-Pet1p60aa-AtPPT1 and pJK148-Pet1p60aa-AtPPT2. In addition, we created a plasmid construct pJK148-Pet1p60aa. Furthermore, the gfp ORF was introduced between the pet1+ promoter and start codon of AtPPTs and Pet1p in pJK148-AtPPT1, pJK148-AtPPT2, pJK148-Pet1p60aa-AtPPT1, and pJK148-Pet1p60aa-AtPPT2 to create the plasmids pJK148-GFP-AtPPT1, pJK 148-GFP-AtPPT2, pJK148-GFP-Pet1p60aa-AtPPT1, and pJK148-GFP-Pet1p60aa-AtPPT2, respectively. The plasmid pAU-Gms1p-RFP was prepared as described previously (Nakase et al., 2010). Fluorescence microscopy
To localize GFP- or RFP-tagged proteins, S. pombe cells expressing these proteins were grown overnight, cells were observed with a Nikon ECLIPSE 80i microscope (Nikon, Japan) and images of cells were captured with a Cool
FEMS Yeast Res 14 (2014) 1101–1109
Asahi Glass, Japan Matsuzawa et al. (2012) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study
SNAP CCD camera using the software METAMORPH (Roper Scientific, San Diego, CA). Alcian blue staining
To analyze negatively-charged glycan on the cell wall of S. pombe, procedures of Alcian blue staining and its data quantitation were performed as described previously with some modifications (Matsuzawa et al., 2012; Yoritsune et al., 2013). To add pyruvate onto Gal residues of oligosaccharides of cell wall, cells (OD600 of 2.0) were collected and washed three-times with water. Thereafter, cells were suspended in 500 lL of reaction solution [100 mM MOPS-NaOH (pH 6.0), 20 mM PEP monopotassium salt and 200 lg of recombinant Pvg1p] and incubated at 30 °C for 1 h, and subsequently, cells were stained using Alcian blue. Recombinant Pvg1p was prepared as described previously (Yoritsune et al., 2013).
Results Pvg1p localizes to the Golgi apparatus
To determine the cellular localization of Pvg1p, we added GFP tag to both the N- and C-termini of Pvg1p and expressed them in the pvg1D strain. We first checked the functionality of these GFP fusion proteins by staining the cells with Alcian blue, which binds to PvGal of S. pombe, and then determined the amount of PvGal on the cell surface (Matsuzawa et al., 2012). We found that the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
1104
Alcian blue stain level (μg per OD600 nm)
(a)
20 18 16 14 12 10 8 6 4 2 0
WT
pvg1Δ
pvg1Δ GFPPvg1p
pvg1Δ Pvg1pGFP
(b) Nomarski
GFP-Pvg1p
Gms1p-RFP
Merge
Fig. 1. Pvg1p localizes to the Golgi apparatus. (a) Alcian blue staining was carried out to determine the amount of PvGal on the cell surface of each indicated strain. The strain indicated here as pvg1D was used as a negative control. Three independent experiments were performed and results shown are mean standard error. (b) Cells expressing both GFP-Pvg1p and Gms1p-RFP were cultured overnight and observed using a fluorescent microscope. Gms1p was used as a marker protein for Golgi. In the merged image, GFP-Pvg1p colocalized with Gms1p-RFP, suggesting that Pvg1p is localized to the Golgi apparatus.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Construction of disruptants of putative PEP transporter genes
As the pyruvyltransferase Pvg1p is localized to the Golgi apparatus, PEP, which is the substrate of the enzyme, should also exist in the organelle. To explore the possibility that a PEP transporter is involved in transporting PEP from the cytoplasm to the Golgi apparatus, we exploited information available on the well-characterized AtPPT1 gene that encoded a PEP transporter in A. thaliana (Fischer et al., 1997; Knappe et al., 2003). By searching the S. pombe genome database (PomBase; http://www. pombase.org/), we identified following three genes that were homologous to AtPPT1: namely, SPAC22F8.04, SPBC83.11, and SPAC22E12.01, and designated them as pet1+, pet2+, and pet3+ (PEP transporter), respectively. The deduced amino acid sequences of Pet1p, Pet2p, and Pet3p exhibited 16%, 17%, and 20% sequence homology to AtPPT1, as determined by analyzing these sequences using CLUSTALW (ver2.1; http://clustalw.ddbj.nig.ac.jp/; Supporting Information, Fig. S1). To examine the function of each gene, we next generated three single gene disruptants, each carrying a disruption in the pet1+, pet2+, or pet3+ gene, and performed alcian blue staining assay to determine the amount of PvGal on the cell surface of each strain. As shown in Fig. 2, cell surface levels of PvGal in pvg1D and pet1D were significantly lower than that in the control cells; on the other hand, cell surface level of PvGal in pet2D was approximately two-thirds of that in the control cells, and cell surface levels of PvGal in pet3D and control cells were same. These results suggested that Pet1p primarily and Pet2p partially contribute to the production of PvGal on the cell surface, possibly by functioning as PEP transporters, whereas Pet3p has almost no role in this process. Therefore, we decided not
Alcian blue stain level (μg per OD600 nm)
parent strain pvg1D had significantly reduced level of PvGal on the cell surface than the control cells (Fig. 1a), as was reported earlier (Yoritsune et al., 2013). Although expression of GFP-Pvg1p (GFP fused to the N-terminal end of Pvg1p) in these cells fully restored the PvGal level on the cell surface, expression of Pvg1p-GFP (GFP fused to the C-terminal end of Pvg1p) failed to do so (Fig. 1a). Therefore, we examined the GFP-Pvg1p expressing cells by fluorescence microscopy to determine the cellular localization of Pvg1p and found that they were expressed as punctate structures (Fig. 1b). To verify whether Pvg1p is localized to the Golgi apparatus, we coexpressed GFPPvg1p and RFP-fused Gms1p, which is a UDP-Gal transporter and used as a Golgi marker protein (Tabuchi et al., 1997; Nakase et al., 2010). As shown in Fig. 1b, GFP-Pvg1p clearly colocalized with Gms1p-RFP. Taken together, these results suggested that Pvg1p is localized to the Golgi apparatus and is functionally active [i.e. adds pyruvate to the N-glycan oligosaccharides, as was demonstrated earlier by Yoritsune et al. (2013)].
20 18 16 14 12 10 8 6 4 2 0
WT
pvg1Δ
pet1Δ
pet2Δ
pet3Δ
pet1Δ pet2Δ
Fig. 2. Analysis of alcian blue stained petD strains. Alcian blue staining was carried out as described in Fig. 1a and the amount of PvGal on the cell surface of each strain was quantified. pvg1D, negative control. Results shown are mean standard error (n = 3).
FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1105
to use the pet3D strain for further analysis. We also constructed a pet1D/pet2D double disruptant strain, surface level of PvGal in which was further reduced than that in the single disruptant pet1D strain, suggesting that Pet1p and Pet2p might be working cooperatively.
and C-terminal GFP fusion constructs of Pet1p and Pet2p in the respective disruptants. All four fusion proteins showed the similar localization pattern (i.e. punctate structures), which indicated that they were probably localized to the Golgi apparatus (Fig. 3a). We also stained these cells with alcian blue and found that both GFP-Pet1p and Pet1p-GFP constructs complemented the phenotypic defect of the pet1D cells, and that the Pet2p-GFP fusion protein fully and GFPPet2p fusion protein partially complemented the phenotypic defect of the pet2D cells (Fig. 3b). Taken together, our results suggested that the GFP fusion constructs of Pet1p and Pet2p,
Both Pet1p and Pet2p localize to the Golgi apparatus
To determine the cellular localization of putative S. pombe PEP transporters Pet1p and Pet2p, we expressed N-terminal (a)
FEMS Yeast Res 14 (2014) 1101–1109
(b) Alcian blue stain level (μg per OD600 nm)
Fig. 3. GFP-fused Pet proteins are functional. (a) Schizosaccharomyces pombe pet1D cells expressing GFP-Pet1p or Pet1p-GFP and pet2D cells expressing GFP-Pet2p or Pet2p-GFP were cultured overnight and then observed using a fluorescent microscope. All four GFP fusion proteins appeared to be expressed as punctate structures. (b) Alcian blue staining of cells and the amount of PvGal on the cell surface of each strain was determined as described under Fig. 1a. Expression of Pet1p and Pet2p (as N- or C-terminal GFP fusion proteins) in S. pombe pet1D and pet2D cells, respectively, using plasmid constructs described in the Materials and methods, restored the PvGal level on the cell surface of each disruptant to that of the control cell level. Three independent experiments were carried out and results shown are mean standard error.
GFP-Pet1p
Nomarski
20 18 16 14 12 10 8 6 4 2 0
Nomarski Pet1p-GFP
Nomarski
GFP-Pet2p
Nomarski
Pet2p-GFP
WT
pet1Δ
pet1Δ pet1Δ pet2Δ GFP- Pet1pPet1p GFP
pet2Δ GFPPet2p
pet2Δ Pet2pGFP
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
1106
generated in this study, are functional, apart from GFPPet2p. To further confirm the localization of Pet1p, we expressed Gms1p-RFP in the Pet1p-GFP expressing strain. As shown in Fig. 4, Pet1p-GFP colocalized with the Gms1p-RFP in dot-like structures. Taken together, these results suggested that Pet1p, which is situated on the membrane of the Golgi apparatus, transports PEP from the cytoplasm into the organelle. Chimeric constructs of Pet1p and AtPPT proteins are able to suppress the phenotype of pet1D cells
So far, we have demonstrated that Pet1p is mainly responsible for the transport of PEP into the Golgi apparatus of S. pombe. To further examine whether Pet1p might function similarly as AtPPT1 and AtPPT2, both of which are well-characterized PEP transporters found in A. thaliana, we individually expressed these two plant PEP transporters in pet1D cells. Resultant strains expressing either full-length AtPPT1 or full-length AtPPT2 could not suppress the phenotype of pet1D cells (data not shown). Indeed, expression of GFP-tagged AtPPT1 and AtPPT2 in S. pombe showed ER-like localization (GFP-AtPPT1, Fig. 5a), probably because of these fusion proteins were not targeted properly from the ER to the Golgi apparatus. Protein sequence analysis using the SOSUI program (http://harrier.nagaha-
Nomarski
Pet1p-GFP
Merge
Gms1p-RFP
Fig. 4. Pet1p localizes to the Golgi apparatus. Gms1p-RFP expressing plasmid construct was introduced into the cells expressing Pet1p-GFP. The resultant cells were cultured overnight and observed using a fluorescent microscope. In the merged image, Pet1p-GFP colocalized with Gms1p-RFP, indicating that Pet1p is localized to the Golgi apparatus.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ma-i-bio.ac.jp/sosui/) revealed that Pet1p, AtPPT1, and AtPPT2 are predicted to contain 10, 7, and 5 transmembrane domains, respectively (Fig. 5b). In addition, each protein is predicted to contain an N-terminal cytoplasmic region. We reasoned that the differences in the N-terminal regions of Pet1p and AtPPTs might be responsible for the mistargeting of AtPPTs in S. pombe. Therefore, to increase the possibility of functional expression of these plant transporters in S. pombe, we replaced the N-terminal portions of the AtPPT1 and AtPPT2 with the N-terminal cytoplasmic region (containing first 60 amino acid residues) of Pet1p (Fig. 5c). We also fused GFP to the N-terminal end of each one of these two chimeric transporters. When expressed in pet1D cells, both GFP-Pet1p60aa-AtPPT1 and GFP-Pet1a60aa-ATPPT2 were expressed as punctate structures (Fig. 5d). Expression of these chimeric proteins also partially suppressed the phenotype of pet1D cells (Fig. 5e). To exclude the possibility that the N-terminal region of Pet1p might be sufficient in complementing the pet1D phenotype, we separately expressed the first 60 aa region of Pet1p in pet1D cells. Our results clearly showed that the expression of only 60 aa of Pet1p could not complement the phenotypic defect in pet1D cells (Fig. 5e), suggesting that rest of the sequence of either AtPPT1 or AtPPT2 protein, but not 60 aa of Pet1p, is sufficient to form a functional protein that could suppress the pet1D phenotype (Fig. 5e). Taken together, these results suggested that Pet1p functions as PEP transporters, just like the plant PEP transporters. Addition of recombinant Pvg1p to living cells can suppress the pet1D phenotype
Previously, we demonstrated that addition of recombinant Pvg1p to living cells could complement the pvg1D phenotype, as judged by alcian blue staining, likely because the addition of Pvg1p from outside could transfer pyruvate onto the Gal residues on the cell wall (Yoritsune et al., 2013). To investigate whether pet1D cells would behave similarly as the pvg1D cells, we repeated the same experiment using the pet1D cells. As shown in Fig. 6, the alcian blue staining level of pet1D cells was significantly increased following incubation with extracellularly added recombinant Pvg1p and PEP, and the increase in staining level was similar for the pet1D and pvg1D cells. This result suggested that the amount of N-linked b-Gal residues, which were surface exposed and not modified by pyruvate, was about same in both pet1D and pvg1D cells.
Discussion To understand the molecular mechanism of N-glycan protein synthesis, it is essential to determine where and FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
1107
Fig. 5. Expressions of chimeric AtPPT proteins suppress the phenotype of the pet1D strain. (a) GFP-tagged AtPPT1 was expressed in the pet1D cells, and cells expressing the GFP fusion protein were observed using a fluorescent microscope. The fusion protein was likely localized to ER-like structures. (b) Transmembrane domains of Pet1p, AtPPT1, and AtPPT2, as predicted by SOSUI program, are shown. (c) Schematic diagram depicting the strategy for constructing the AtPPT1 chimera, where the N-terminal portion of AtPPT1 was replaced with the N-terminal portion (first 60 amino acid residues) of Pet1p. (d) GFP-tagged Pet1p60aa-AtPPT1 and Pet1p60aa-AtPPT2 chimeric proteins were individually expressed in the pet1D cells, and cells expressing the GFP fusion protein were observed using a fluorescent microscope. Both chimeric proteins appeared to be expressed as Golgi-like punctate structures. (e) Alcian blue staining and quantitation of PvGal on the cell surface of each strain was carried out as described before. Note that the expression of AtPPT1 or AtPPT2 chimeric construct partially suppressed the phenotype of the pet1D cells. However, complementation of phenotype was not observed in cells expressing only the first 60 amino acid residues of Pet1p. Three independent experiments were performed and results shown are mean standard error.
20 18 16 14 12 10 8 6 4 2 0 WT
(b)
pet1Δ
pet1Δ chimeric AtPPT1
AtPPT1
Pet1p
(c)
Pet1p
(d) Nomarski
AtPPT1
GFP-chimeric AtPPT1
pet1Δ chimeric AtPPT2
pet1Δ Pet1p 60 aa
AtPPT2
80 aa
100 aa
how oligosaccharide-modifying enzymes are regulated. In fission yeast, negative charge conferred by PvGal moieties of oligosaccharides is a major determinant of physiological cell response (Gemmill & Trimble, 1996; Tanaka & Takegawa, 2001; Matsuzawa et al., 2011). We have recently characterized the pyruvyltransferase Pvg1p; however, at that time, we did not examine where the enzyme was localized (Yoritsune et al., 2013). Therefore, at the beginning of this study, we expressed a functionally active, N-terminally GFP-tagged Pvg1p in S. pombe and found using fluorescence microscopy that this fusion protein was localized to the Golgi apparatus. Previously, Pvg1p was shown to be localized in the periplasmic space (Andreishcheva et al., 2004). In that study, however, a C-terminally GFP-tagged Pvg1p was used and improper fusion might have lead to its mislocalization. Thus, from our observation, we concluded that Pvg1p is localized to
FEMS Yeast Res 14 (2014) 1101–1109
(e) Alcian blue stain level (μg per OD600 nm)
(a) GFP-AtPPT1
Nomarski
Chimeric AtPPT1
GFP-chimeric AtPPT2
the Golgi apparatus, where it attaches pyruvate to the Gal residues of oligosaccharides. As PEP, from which pyruvate is produced, should be supplied to the lumen of the Golgi apparatus, we speculated that there ought to be transporter(s) for delivering the cytoplasmic PEP into the organelle. Based on the results of BLAST search against the A. thaliana PEP transporter AtPPT1, we found three possible candidate genes in S. pombe. According to the S. pombe genome database, Pet1p and Pet2p were predicted to be triose phosphate transporters, whereas Pet3p showed highest homology to a probable sugar phosphate/phosphate translocator in A. thaliana (NP_172135.2) with an e-value of 7e53. Indeed, results described in this study showed that Pet3p had no role in the modification of Gal to PvGal on the cell surface. On the other hand, Pet1p and Pet2p may play primary and secondary roles in producing PvGal on ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
K.-i. Yoritsune et al.
Alcian blue stain level (μg per OD600 nm)
1108
20 18 16 14 12 10 8 6 4 2 0
Strain
WT
WT
pvg1 Δ pvg1 Δ pet1 Δ
pet1 Δ
Pvg1p Fig. 6. Addition of recombinant Pvg1p suppresses the phenotype of the pet1D strain. Cultures of indicated strains were incubated with or without recombinant Pvg1p and PEP at 30 °C for 1 h, and cells were then stained with alcian blue and the amount of PvGal on the cell surface was determined as before. Note that the addition of recombinant Pvg1p and PEP to the living pet1D cells increased the amount of PvGal on the cell surface. The strain pvg1D was used here as a control. Three independent experiments were performed and results shown are mean standard error.
the cell surface, respectively. In S. cerevisiae, negative charge of the cell surface is provided by mannosylphosphate, unlike S. pombe, and thus, it is not necessary for PEP to be transported into the Golgi apparatus. Consistent with this, in budding yeast, there is no ortholog for Pet1p and one for Pet2p with relatively low homology (YJL193W; e-value, 6.6e22). In other fungi, we found that the filamentous fungus Trichoderma reesei has the Pet1p (XP_006969429.1; e-value, 8e47), Pet2p (XP_0069 68103.1; e-value, 5e90), and Pvg1p (XP_006969404.1; e-value, 3e45) orthologs. Therefore, it is intriguing to investigate whether pyruvylated oligosaccharides exist in T. reesei like S. pombe. Analyses of substrate specificities of AtPPT1 and AtPPT2 showed that AtPPT1 preferred 2-phosphoglycerate and PEP over 3-phosphoglycerate as a substrate and AtPPT2 preferred PEP over 2-phosphoglycerate and 3-phosphoglycerate as a substrate (Knappe et al., 2003). Hence, it is possible that Pet1p and Pet2p in S. pombe may have different substrate specificities. Based on results described in this study, we believe that Pet1p has more preference for PEP as a substrate than Pet2p. Moreover, as we could not find any phenotype for the pet3D cells, Pet3p might be transporting a different substrate, such as a sugar phosphate, as was predicted by the BLAST search. We undertook to functionally express GFP-AtPPT fusion proteins in S. pombe, but initially these fusion proteins did not reach the Golgi apparatus and instead they ended up in the ER-like structures, likely because of unsuccessful targeting. In plant, both AtPPTs are localized in the chloroplast (Knappe et al., 2003); thus, they
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
probably lacked the signal for localizing to the Golgi apparatus in S. pombe. To assist the AtPPTs to reach the Golgi apparatus in fission yeast, we next replaced the N-terminal cytoplasmic regions of AtPPT1 and AtPPT2 with the first 60 aa of Pet1p. These chimeric constructs successfully reached the Golgi apparatus, suggesting that the N-terminal part of Pet1p contained the signal for Golgi localization. Although recent protein sequence analysis softwares could better predict the subcellular localization of proteins, predicting the localization of transmembrane proteins is still difficult, especially for the ones that are localized to the Golgi apparatus (Mukai et al., 2011). Thus, more detailed analyses of Pet1p and Pet2p may provide appropriate information regarding the Golgi localization sequence. In summary, we examined three putative S. pombe PEP transporter genes, one of which (i.e. pet3+) probably does not have the function of PEP transport. We demonstrated that Pet1p is mainly responsible for transporting PEP from the cytoplasm into the Golgi apparatus of S. pombe, and Pet2p may have a supporting role in PEP transport. In conjunction with the enzymatic characterization of Pvg1p that was carried out earlier, we have found out how pyruvate is attached to Gal residues of oligosaccharides. Further analysis of PvGal, including the structural analysis of Pvg1p, will be pursued to gain new insights in the field of glycobiology that could find applications in developing novel pharmaceutical agents.
Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (K.T.). The authors declare that they have no conflict of interest.
References Alfa C, Fantes P, Hyams J, McLeod M & Warbrick E (1993) Experiments with Fission Yeast. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Andreishcheva EN, Kunkel JP, Gemmill TR & Trimble RB (2004) Five genes involved in biosynthesis of the pyruvylated Galb1,3-epitope in Schizosaccharomyces pombe N-linked glycans. J Biol Chem 279: 35644–35655. Fischer K, Kammerer B, Gutensohn M, Arbinger B, Weber A, H€ausler RE & Fl€ ugge UI (1997) A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/ phosphate antiporter. Plant Cell 9: 453–462. Gemmill TR & Trimble RB (1996) Schizosaccharomyces pombe produces novel pyruvate-containing N-linked oligosaccharides. J Biol Chem 271: 25945–25949.
FEMS Yeast Res 14 (2014) 1101–1109
Analysis of S. pombe phosphoenolpyruvate transporters
Gemmill TR & Trimble RB (1998) All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4,6-O-[(R)-(1-carboxyethylidine)]-Galb1,3Gala1. Glycobiology 8: 1087–1095. Gemmill TR & Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426: 227–237. Knappe S, L€ ottgert T, Schneider A, Voll L, Fl€ ugge UI & Fischer K (2003) Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis – AtPPT1 may be involved in the provision of signals for correct mesophyll development. Plant J 36: 411– 420. Matsuzawa T, Morita T, Tanaka N, Tohda H & Takegawa K (2011) Identification of a galactose-specific flocculin essential for non-sexual flocculation and filamentous growth in Schizosaccharomyces pombe. Mol Microbiol 82: 1531–1544. Matsuzawa T, Yoritsune K & Takegawa K (2012) MADS box transcription factor Mbx2/Pvg4 regulates invasive growth and flocculation by inducing gsf2+ expression in fission yeast. Eukaryot Cell 11: 151–158. Mukai Y, Yoshizawa M, Sasaki T, Ikeda M, Tomii K, Hirokawa T & Suwa M (2011) Discrimination of Golgi type II membrane proteins based on their hydropathy profiles and the amino acid propensities of their transmembrane regions. Biosci Biotechnol Biochem 75: 82–88. Nakase M, Tani M, Morita T, Kitamoto HK, Kashiwazaki J, Nakamura T, Hosomi A, Tanaka N & Takegawa K (2010) Mannosylinositol phosphorylceramide is a major sphingolipid component and is required for proper localization of plasma-membrane proteins in Schizosaccharomyces pombe. J Cell Sci 123: 1578–1587.
FEMS Yeast Res 14 (2014) 1101–1109
1109
Odani T, Shimma Y, Wang XH & Jigami Y (1997) Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett 420: 186–190. Parolis LA, Duus JO, Parolis H, Meldal M & Bock K (1996) The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the structure of the phosphomannan backbone. Carbohydr Res 293: 101–117. Spillmann D, Hard K, Thomas-Oates J, Vliegenthart JF, Misevic G, Burger MM & Finne J (1993) Characterization of a novel pyruvylated carbohydrate unit implicated in the cell aggregation of the marine sponge Microciona prolifera. J Biol Chem 268: 13378–13387. Tabuchi M, Tanaka N, Iwahara S & Takegawa K (1997) The Schizosaccharomyces pombe gms1+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem Biophys Res Commun 232: 121–125. Tanaka N & Takegawa K (2001) Biosynthetic pathway and physiological role of galactose-containing oligosaccharides in fission yeast Schizosaccharomyces pombe. Trends Glycosci Glycotechnol 13: 519–532. Yoritsune K, Matsuzawa T, Ohashi T & Takegawa T (2013) The fission yeast Pvg1p has galactose-specific pyruvyltransferase activity. FEBS Lett 587: 917–921.
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Amino acid sequence alignments of Pet1p, Pet2p, Pet3p, and AtPPT1.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
