Commentary
Glycosylphosphatidylinositol Anchoring: Control through Modification Since their discovery (Low and Saltiel, 1988), glycosylphosphatidylinositol-anchored proteins (GPI-APs) have provoked intense interest as crucial regulators for growth, morphogenesis, reproduction, and disease pathogenesis in organisms ranging from yeast and trypanosomes to animals and plants. The lipid moiety, the glycosylphosphatidylinositol (GPI) anchor, is synthesized in the endoplasmic reticulum (ER); the protein component is cotranslationally inserted into the ER and posttranslationally modified by the addition of a GPI anchor (Kinoshita et al., 2013; Fig. 1). GPI-APs are then transported via the Golgi to the outer surface of the plasma membrane. The lipid anchor mediates stable attachment of these proteins to the cell surface, where some play important roles as signaling regulators from sphingolipid- and sterol-enriched membrane microdomains (Simons and Gerl, 2010). Some GPI-APs are released from the cell membrane by phosphatidylinositol-specific phospholipases to the extracellular matrix, where they might engage in processes such as cell adhesion and cell-cell communication. In Arabidopsis (Arabidopsis thaliana), there are about 250 predicted GPI-APs (Borner et al., 2003), a relatively large number compared with about 150 in mammals and 50 in the budding yeast (Saccharomyces cerevisiae). Important functions for plant GPI-APs have been elucidated through the study of individual proteins, such as the COBRA family in cell expansion and cell wall biosynthesis (Brady et al., 2007), ARABINOGALACTAN PROTEIN18 in megagametogenesis (Demesa-Arévalo and Vielle-Calzada, 2013), and LORELEI in the pollen tube-female gametophyte interaction (Capron et al., 2008; Tsukamoto et al., 2010; Duan et al., 2014). However, it is the studies of mutants defective in GPI biosynthesis that underscore the general importance of GPI-APs as a class: lacking the capacity to assemble the anchor is lethal. The GPI anchor is synthesized by an elaborate biosynthetic pathway, starting on the cytoplasmic side of the ER and ending with a completely assembled core anchor on the lumenal surface of the ER (Fig. 1). Prior to their transport out of the ER, proteins destined for GPI modification are cleaved at a C-terminal signature sequence by a GPI transamidase complex that in two enzymatic steps concomitantly attaches a GPI anchor to the C terminus of processed proteins (Kinoshita, 2014). Most of the knowledge on GPI biosynthesis and GPI-AP modification is derived from studies in mammals and yeast, but the pathway is likely to be conserved in plants (Ellis et al., 2010). In a recent article in Plant Physiology, Dai et al. (2014) reported that a GPI Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.114.246926 748
anchor biosynthesis mutant, abnormal pollen tube guidance1 (atpg1), displays both embryo lethality and severely depressed male fertility. They determined that APTG1 is homologous to the yeast GPI10 and human PIG-B (for phosphatidylinositol glycan anchor biosynthesis) proteins, the last of three distinct mannosyltransferases that modify the precursor anchor (Fig. 1), and showed that APTG1 can functionally substitute for GPI10 in a conditionally lethal gpi10 yeast mutant. Previous studies have demonstrated that Arabidopsis SETH1 (a male fertility god in Egyptian mythology), SETH2, and PEANUT1 (PNT1), encoding homologs of mammalian PIG-C, PIG-A, and PIG-M (Fig. 1) and their corresponding yeast counterparts, respectively, are important for male fertility (Lalanne et al., 2004; Gillmor et al., 2005). In addition, loss of the first mannosyltransferase in the pathway in pnt1 results in early seedling lethality. pnt1 embryos are severely defective, displaying various cell division anomalies and exhibiting altered levels and ectopic deposition of cell wall polymers. The results reported by Dai et al. (2014), therefore, further demonstrate
Figure 1. GPI anchor biosynthesis pathway. Ten or 11 stepwise modifications of phosphoinositide occur, starting from the synthesis of N-glucosamine-phosphoinositide on the cytoplasmic surface of the ER, followed by its flipping to the ER lumenal side for additional modifications, ending with the addition of the terminal ethanolamine phosphate. Proteins destined for GPI modification are synthesized with a C-terminal signature sequence recognized by the GPI transamidase (a five-protein-enzyme complex) that concomitantly cleaves the peptide at what is designated as the v and v+1 amino acids and attaches the GPI anchor in a transamination reaction (red arrows). The GPI-modified proteins are then sorted and transported via the Golgi apparatus to the cell membrane. The established biosynthetic proteins from Arabidopsis and their mammalian homologs are indicated; the galactosylation step appears to be plant specific. The diagram is modeled after figure 3 in Ellis et al. (2010), which also provided a complete list of potential plant orthologs of the human and yeast proteins in the pathway.
Plant PhysiologyÒ, October 2014, Vol. 166, pp. 748–750, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved.
Commentary
imaging at the cell surface and biochemical approaches to determine the constituents in these membrane microdomains (Simons and Gerl, 2010) should accelerate our understanding of the importance of GPI anchoring as a conserved strategy among eukaryotes to control a wide range of processes. Alice Y. Cheung Department of Biochemistry and Molecular Biology, Molecular and Cellular Biology Program, and Plant Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts 01003 ORCID ID: 0000-0002-7973-022X
Figure 2. Pollen tube targeting of ovules in an Arabidopsis pistil. GUSexpressing pollen grains pollinated the pistil. Each blue dot represents discharged cytoplasm from a pollen tube that, in response to attractants, has successfully targeted the ovule and penetrated the female gametophyte and was induced to burst. The cytoplasmic discharge releases sperm for fertilization.
the conservation of the GPI biosynthesis pathway and the importance of GPI anchoring in plant development and reproduction. The aptg1 mutant was isolated in a search for mutants defective in pollen tube targeting of ovules (Fig. 2), an intriguing and crucial step in plant reproduction. A pollen tube is guided to an ovule by attractants, and upon reaching the target, the female gametophyte, the pollen tube ruptures, ejecting its cytoplasm and releasing sperm for fertilization (Dresselhaus and Franklin-Tong, 2013). aptg1 pollen tubes either fail to target ovules or undertake a more twisted pathway before entering an ovule. In an earlier study, Li et al. (2013) showed that a GPI-AP, COBRA-LIKE10 (COBL10), is required to maintain normal pollen tube growth rates and ovule targeting efficiency. In aptg1 pollen tubes, citrine fluorescent protein-COBL10 was absent from its normal apical membrane location while the citrine fluorescent signal in the cytoplasm was more intense, implying that the diminished presence of COBL10 on the apical membrane could be an underlying cause for the ovule-targeting phenotype. This observation also demonstrates that GPI anchoring is important for the subsequent sorting and transport of these proteins to their destined locations (Kinoshita et al., 2013) and consistent with a wholesale failure of GPI-APs to reach their functional locations as underlying lethality in GPI biosynthesis mutants. While it is clear that major biological roles are played by GPI-APs, many questions remain. Most constituents of the plant GPI anchor biosynthetic pathway remain to be functionally established (Fig. 1). Much has been said about the membrane environments where GPI-APs are localized, but we are far from understanding the precise roles they play in assembling these domains and regulating their functional dynamics. Advances in high-resolution Plant Physiol. Vol. 166, 2014
Chao Li Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Yan-jiao Zou Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Hen-Ming Wu Department of Biochemistry and Molecular Biology and Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Massachusetts 01003 ORCID ID: 0000-0003-2108-3848 ACKNOWLEDGMENTS We thank Sheila McCormick (University of California, Berkeley) and Dong Wang (University of Massachusetts) for comments on the article.
LITERATURE CITED Borner GHH, Lilley KS, Stevens TJ, Dupree P (2003) Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis: a proteomic and genomic analysis. Plant Physiol 132: 568–577 Brady SM, Song S, Dhugga KS, Rafalski JA, Benfey PN (2007) Combining expression and comparative evolutionary analysis: the COBRA gene family. Plant Physiol 143: 172–187 Capron A, Gourgues M, Neiva LS, Faure JE, Berger F, Pagnussat G, Krishnan A, Alvarez-Mejia C, Vielle-Calzada JP, Lee YR, et al (2008) Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene. Plant Cell 20: 3038–3049 Dai XR, Gao XQ, Chen GH, Tang LL, Wang H, Zhang XS (2014) ABNORMAL POLLEN TUBE GUIDANCE1, an endoplasmic reticulum-localized mannosyltransferase homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in human, is required for Arabidopsis pollen tube micropylar guidance and embryo development. Plant Physiol 165: 1544–1556 Demesa-Arévalo E, Vielle-Calzada JP (2013) The classical arabinogalactan protein AGP18 mediates megaspore selection in Arabidopsis. Plant Cell 25: 1274–1287 Dresselhaus T, Franklin-Tong N (2013) Male-female crosstalk during pollen germination, tube growth and guidance, and double fertilization. Mol Plant 6: 1018–1036 Duan Q, Kita D, Johnson EA, Aggarwal M, Gates L, Wu HM, Cheung AY (2014) Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nat Commun 5: 3129 749
Commentary
Ellis M, Egelund J, Schultz CJ, Bacic A (2010) Arabinogalactan-proteins: key regulators at the cell surface? Plant Physiol 153: 403–419 Gillmor CS, Lukowitz W, Brininstool G, Sedbrook JC, Hamann T, Poindexter P, Somerville C (2005) Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis. Plant Cell 17: 1128–1140 Kinoshita T (2014) Enzymatic mechanism of GPI anchor attachment clarified. Cell Cycle 13: 1838–1839 Kinoshita T, Maeda Y, Fujita M (2013) Transport of glycosylphosphatidylinositolanchored proteins from the endoplasmic reticulum. Biochim Biophys Acta 1833: 2473–2478 Lalanne E, Honys D, Johnson A, Borner GHH, Lilley KS, Dupree P, Grossniklaus U, Twell D (2004) SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are
750
required for pollen germination and tube growth in Arabidopsis. Plant Cell 16: 229–240 Li S, Ge FR, Xu M, Zhao XY, Huang GQ, Zhou LZ, Wang JG, Kombrink A, McCormick S, Zhang XS, et al (2013) Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes. Plant J 74: 486–497 Low MG, Saltiel AR (1988) Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 239: 268–275 Simons K, Gerl MJ (2010) Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11: 688–699 Tsukamoto T, Qin Y, Huang Y, Dunatunga D, Palanivelu R (2010) A role for LORELEI, a putative glycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J 62: 571–588
Plant Physiol. Vol. 166, 2014
Glycosylphosphatidylinositol Anchoring: Control through Modification Since their discovery (Low and Saltiel, 1988), glycosylphosphatidylinositol-anchored proteins (GPI-APs) have provoked intense interest as crucial regulators for growth, morphogenesis, reproduction, and disease pathogenesis in organisms ranging from yeast and trypanosomes to animals and plants. The lipid moiety, the glycosylphosphatidylinositol (GPI) anchor, is synthesized in the endoplasmic reticulum (ER); the protein component is cotranslationally inserted into the ER and posttranslationally modified by the addition of a GPI anchor (Kinoshita et al., 2013; Fig. 1). GPI-APs are then transported via the Golgi to the outer surface of the plasma membrane. The lipid anchor mediates stable attachment of these proteins to the cell surface, where some play important roles as signaling regulators from sphingolipid- and sterol-enriched membrane microdomains (Simons and Gerl, 2010). Some GPI-APs are released from the cell membrane by phosphatidylinositol-specific phospholipases to the extracellular matrix, where they might engage in processes such as cell adhesion and cell-cell communication. In Arabidopsis (Arabidopsis thaliana), there are about 250 predicted GPI-APs (Borner et al., 2003), a relatively large number compared with about 150 in mammals and 50 in the budding yeast (Saccharomyces cerevisiae). Important functions for plant GPI-APs have been elucidated through the study of individual proteins, such as the COBRA family in cell expansion and cell wall biosynthesis (Brady et al., 2007), ARABINOGALACTAN PROTEIN18 in megagametogenesis (Demesa-Arévalo and Vielle-Calzada, 2013), and LORELEI in the pollen tube-female gametophyte interaction (Capron et al., 2008; Tsukamoto et al., 2010; Duan et al., 2014). However, it is the studies of mutants defective in GPI biosynthesis that underscore the general importance of GPI-APs as a class: lacking the capacity to assemble the anchor is lethal. The GPI anchor is synthesized by an elaborate biosynthetic pathway, starting on the cytoplasmic side of the ER and ending with a completely assembled core anchor on the lumenal surface of the ER (Fig. 1). Prior to their transport out of the ER, proteins destined for GPI modification are cleaved at a C-terminal signature sequence by a GPI transamidase complex that in two enzymatic steps concomitantly attaches a GPI anchor to the C terminus of processed proteins (Kinoshita, 2014). Most of the knowledge on GPI biosynthesis and GPI-AP modification is derived from studies in mammals and yeast, but the pathway is likely to be conserved in plants (Ellis et al., 2010). In a recent article in Plant Physiology, Dai et al. (2014) reported that a GPI Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.114.246926 748
anchor biosynthesis mutant, abnormal pollen tube guidance1 (atpg1), displays both embryo lethality and severely depressed male fertility. They determined that APTG1 is homologous to the yeast GPI10 and human PIG-B (for phosphatidylinositol glycan anchor biosynthesis) proteins, the last of three distinct mannosyltransferases that modify the precursor anchor (Fig. 1), and showed that APTG1 can functionally substitute for GPI10 in a conditionally lethal gpi10 yeast mutant. Previous studies have demonstrated that Arabidopsis SETH1 (a male fertility god in Egyptian mythology), SETH2, and PEANUT1 (PNT1), encoding homologs of mammalian PIG-C, PIG-A, and PIG-M (Fig. 1) and their corresponding yeast counterparts, respectively, are important for male fertility (Lalanne et al., 2004; Gillmor et al., 2005). In addition, loss of the first mannosyltransferase in the pathway in pnt1 results in early seedling lethality. pnt1 embryos are severely defective, displaying various cell division anomalies and exhibiting altered levels and ectopic deposition of cell wall polymers. The results reported by Dai et al. (2014), therefore, further demonstrate
Figure 1. GPI anchor biosynthesis pathway. Ten or 11 stepwise modifications of phosphoinositide occur, starting from the synthesis of N-glucosamine-phosphoinositide on the cytoplasmic surface of the ER, followed by its flipping to the ER lumenal side for additional modifications, ending with the addition of the terminal ethanolamine phosphate. Proteins destined for GPI modification are synthesized with a C-terminal signature sequence recognized by the GPI transamidase (a five-protein-enzyme complex) that concomitantly cleaves the peptide at what is designated as the v and v+1 amino acids and attaches the GPI anchor in a transamination reaction (red arrows). The GPI-modified proteins are then sorted and transported via the Golgi apparatus to the cell membrane. The established biosynthetic proteins from Arabidopsis and their mammalian homologs are indicated; the galactosylation step appears to be plant specific. The diagram is modeled after figure 3 in Ellis et al. (2010), which also provided a complete list of potential plant orthologs of the human and yeast proteins in the pathway.
Plant PhysiologyÒ, October 2014, Vol. 166, pp. 748–750, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved.
Commentary
imaging at the cell surface and biochemical approaches to determine the constituents in these membrane microdomains (Simons and Gerl, 2010) should accelerate our understanding of the importance of GPI anchoring as a conserved strategy among eukaryotes to control a wide range of processes. Alice Y. Cheung Department of Biochemistry and Molecular Biology, Molecular and Cellular Biology Program, and Plant Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts 01003 ORCID ID: 0000-0002-7973-022X
Figure 2. Pollen tube targeting of ovules in an Arabidopsis pistil. GUSexpressing pollen grains pollinated the pistil. Each blue dot represents discharged cytoplasm from a pollen tube that, in response to attractants, has successfully targeted the ovule and penetrated the female gametophyte and was induced to burst. The cytoplasmic discharge releases sperm for fertilization.
the conservation of the GPI biosynthesis pathway and the importance of GPI anchoring in plant development and reproduction. The aptg1 mutant was isolated in a search for mutants defective in pollen tube targeting of ovules (Fig. 2), an intriguing and crucial step in plant reproduction. A pollen tube is guided to an ovule by attractants, and upon reaching the target, the female gametophyte, the pollen tube ruptures, ejecting its cytoplasm and releasing sperm for fertilization (Dresselhaus and Franklin-Tong, 2013). aptg1 pollen tubes either fail to target ovules or undertake a more twisted pathway before entering an ovule. In an earlier study, Li et al. (2013) showed that a GPI-AP, COBRA-LIKE10 (COBL10), is required to maintain normal pollen tube growth rates and ovule targeting efficiency. In aptg1 pollen tubes, citrine fluorescent protein-COBL10 was absent from its normal apical membrane location while the citrine fluorescent signal in the cytoplasm was more intense, implying that the diminished presence of COBL10 on the apical membrane could be an underlying cause for the ovule-targeting phenotype. This observation also demonstrates that GPI anchoring is important for the subsequent sorting and transport of these proteins to their destined locations (Kinoshita et al., 2013) and consistent with a wholesale failure of GPI-APs to reach their functional locations as underlying lethality in GPI biosynthesis mutants. While it is clear that major biological roles are played by GPI-APs, many questions remain. Most constituents of the plant GPI anchor biosynthetic pathway remain to be functionally established (Fig. 1). Much has been said about the membrane environments where GPI-APs are localized, but we are far from understanding the precise roles they play in assembling these domains and regulating their functional dynamics. Advances in high-resolution Plant Physiol. Vol. 166, 2014
Chao Li Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Yan-jiao Zou Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Hen-Ming Wu Department of Biochemistry and Molecular Biology and Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Massachusetts 01003 ORCID ID: 0000-0003-2108-3848 ACKNOWLEDGMENTS We thank Sheila McCormick (University of California, Berkeley) and Dong Wang (University of Massachusetts) for comments on the article.
LITERATURE CITED Borner GHH, Lilley KS, Stevens TJ, Dupree P (2003) Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis: a proteomic and genomic analysis. Plant Physiol 132: 568–577 Brady SM, Song S, Dhugga KS, Rafalski JA, Benfey PN (2007) Combining expression and comparative evolutionary analysis: the COBRA gene family. Plant Physiol 143: 172–187 Capron A, Gourgues M, Neiva LS, Faure JE, Berger F, Pagnussat G, Krishnan A, Alvarez-Mejia C, Vielle-Calzada JP, Lee YR, et al (2008) Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene. Plant Cell 20: 3038–3049 Dai XR, Gao XQ, Chen GH, Tang LL, Wang H, Zhang XS (2014) ABNORMAL POLLEN TUBE GUIDANCE1, an endoplasmic reticulum-localized mannosyltransferase homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in human, is required for Arabidopsis pollen tube micropylar guidance and embryo development. Plant Physiol 165: 1544–1556 Demesa-Arévalo E, Vielle-Calzada JP (2013) The classical arabinogalactan protein AGP18 mediates megaspore selection in Arabidopsis. Plant Cell 25: 1274–1287 Dresselhaus T, Franklin-Tong N (2013) Male-female crosstalk during pollen germination, tube growth and guidance, and double fertilization. Mol Plant 6: 1018–1036 Duan Q, Kita D, Johnson EA, Aggarwal M, Gates L, Wu HM, Cheung AY (2014) Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nat Commun 5: 3129 749
Commentary
Ellis M, Egelund J, Schultz CJ, Bacic A (2010) Arabinogalactan-proteins: key regulators at the cell surface? Plant Physiol 153: 403–419 Gillmor CS, Lukowitz W, Brininstool G, Sedbrook JC, Hamann T, Poindexter P, Somerville C (2005) Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis. Plant Cell 17: 1128–1140 Kinoshita T (2014) Enzymatic mechanism of GPI anchor attachment clarified. Cell Cycle 13: 1838–1839 Kinoshita T, Maeda Y, Fujita M (2013) Transport of glycosylphosphatidylinositolanchored proteins from the endoplasmic reticulum. Biochim Biophys Acta 1833: 2473–2478 Lalanne E, Honys D, Johnson A, Borner GHH, Lilley KS, Dupree P, Grossniklaus U, Twell D (2004) SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are
750
required for pollen germination and tube growth in Arabidopsis. Plant Cell 16: 229–240 Li S, Ge FR, Xu M, Zhao XY, Huang GQ, Zhou LZ, Wang JG, Kombrink A, McCormick S, Zhang XS, et al (2013) Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes. Plant J 74: 486–497 Low MG, Saltiel AR (1988) Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 239: 268–275 Simons K, Gerl MJ (2010) Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11: 688–699 Tsukamoto T, Qin Y, Huang Y, Dunatunga D, Palanivelu R (2010) A role for LORELEI, a putative glycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J 62: 571–588
Plant Physiol. Vol. 166, 2014
