A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Abstract

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16–18 °C but died within several days after transfer to 28 °C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (Kcat/Km). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype. Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16–18 °C but died within several days after transfer to 28 °C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (Kcat/Km). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype. Phosphomannomutases (PMMs) 8The abbreviations used are:PMMphosphomannomutaseAFLPamplified fragment length polymorphismAsAascorbic acidConAconcanavalin AEMSethyl methanesulfonateGMPPGDP-d-mannose pyrophosphorylaseGPIglycosylphosphatidylinositolInDelinsertion/deletionMSMurashige and SkoogPDIprotein-disulfide isomerasetstemperature-sensitiveBCback-crossedMbmegabase(s).8The abbreviations used are:PMMphosphomannomutaseAFLPamplified fragment length polymorphismAsAascorbic acidConAconcanavalin AEMSethyl methanesulfonateGMPPGDP-d-mannose pyrophosphorylaseGPIglycosylphosphatidylinositolInDelinsertion/deletionMSMurashige and SkoogPDIprotein-disulfide isomerasetstemperature-sensitiveBCback-crossedMbmegabase(s). catalyze the interconversion of mannose 6-phosphate and mannose 1-phosphate and are required for the synthesis of GDP-mannose (Fig. 1). Both prokaryotes and eukaryotes utilize GDP-mannose in the synthesis of complex structural carbohydrates. Accordingly, this sugar nucleotide contributes to the synthesis of different structural carbohydrates in plant cell walls (1Seifert G.J. Curr. Opin. Plant Biol. 2004; 7: 277-284Crossref PubMed Scopus (257) Google Scholar) and fulfills a key role in the biosynthesis of ascorbic acid (AsA) in plants (2Wheeler G.L. Jones M.A. Smirnoff N. Nature. 1998; 393: 365-369Crossref PubMed Scopus (795) Google Scholar, 3Qian W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). GDP-mannose is also essential for post-translational modifications, such as protein glycosylation and glycosylphosphatidylinositol (GPI) anchoring in eukaryotes, because mannose, derived from GDP-mannose, is a crucial building block of the core glycan chain attached to the modified proteins (4Lerouge P. Cabanes-Macheteau M. Rayon C. Fischette-Lainé A-C. Gomord V. Faye L. Plant Mol. Biol. 1998; 38: 31-48Crossref PubMed Google Scholar, 5Spiro R.G. Glycobiology. 2002; 12: 43R-56RCrossref PubMed Scopus (999) Google Scholar). phosphomannomutase amplified fragment length polymorphism ascorbic acid concanavalin A ethyl methanesulfonate GDP-d-mannose pyrophosphorylase glycosylphosphatidylinositol insertion/deletion Murashige and Skoog protein-disulfide isomerase temperature-sensitive back-crossed megabase(s). phosphomannomutase amplified fragment length polymorphism ascorbic acid concanavalin A ethyl methanesulfonate GDP-d-mannose pyrophosphorylase glycosylphosphatidylinositol insertion/deletion Murashige and Skoog protein-disulfide isomerase temperature-sensitive back-crossed megabase(s). Because of their involvement in such fundamental processes, mutations in PMM genes often lead to severe and pleiotropic phenotypes. In the yeast Saccharomyces cerevisiae, a temperature-sensitive (ts) mutant affected in the PMM homolog SEC53 is arrested in growth at restrictive temperature. In fact, SEC53 has been classified as an essential gene because of the lethal phenotype of its null mutant (6Bernstein M. Hoffmann W. Ammerer G. Schekman R. J. Cell Biol. 1985; 101: 2374-2382Crossref PubMed Scopus (35) Google Scholar, 7Kepes F. Schekman R. J. Biol. Chem. 1988; 263: 9155-9161Abstract Full Text PDF PubMed Google Scholar). Furthermore, PMM gene deletion mutants of the protozoan parasite Leishmania mexicana remain viable but are no longer able to establish an infection (8Garami A. Mehlert A. Ilg T. Mol. Cell. Biol. 2001; 21: 8168-8183Crossref PubMed Scopus (79) Google Scholar). In mouse, disruption of the PMM2 gene causes early embryonic lethality (9Thiel C. Lübke T. Matthijs G. von Figura K. Körner C. Mol. Cell. Biol. 2006; 26: 5615-5620Crossref PubMed Scopus (59) Google Scholar), and in humans mutations in PMM2 lie at the basis of Jaeken syndrome (also termed carbohydrate-deficient glycoprotein syndrome type Ia), a severe clinical disorder that provokes impaired neurological development and increased childhood mortality (10Van Schaftingen E. Jaeken J. FEBS Lett. 1995; 377: 318-320Crossref PubMed Scopus (306) Google Scholar). In both S. cerevisiae and humans, defects in protein glycosylation due to lack of GDP-mannose have been pinpointed as the underlying cause for the observed phenotypes (7Kepes F. Schekman R. J. Biol. Chem. 1988; 263: 9155-9161Abstract Full Text PDF PubMed Google Scholar, 10Van Schaftingen E. Jaeken J. FEBS Lett. 1995; 377: 318-320Crossref PubMed Scopus (306) Google Scholar). The AsA deficiency of the Arabidopsis thaliana vitamin c1 (vtc1) mutant demonstrated that GDP-mannose is also involved in the biosynthesis of AsA (11Conklin P.L. Norris S.R. Wheeler G.L. Williams E.H. Smirnoff N. Last R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4198-4203Crossref PubMed Scopus (302) Google Scholar). The VTC1 locus encodes a GDP-mannose pyrophosphorylase (GMPP), the enzyme that produces GDP-mannose from mannose 1-phosphate and acts immediately downstream of PMM (Fig. 1). As a consequence, vtc1 plants contain only ∼25% of wild type AsA levels (11Conklin P.L. Norris S.R. Wheeler G.L. Williams E.H. Smirnoff N. Last R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4198-4203Crossref PubMed Scopus (302) Google Scholar, 12Conklin P.L. Pallanca J.E. Last R.L. Smirnoff N. Plant Physiol. 1997; 115: 1277-1285Crossref PubMed Scopus (166) Google Scholar). Very recently, has been for the role of PMM in AsA biosynthesis in plants W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). of PMM levels gene a in AsA in PMM levels in an in AsA of PMM in Arabidopsis also increased AsA no Arabidopsis mutants were and to plants were that the PMM gene is This to plants decreased PMM levels the involvement of PMM in W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). we an ethyl methanesulfonate of Arabidopsis in for mutations plant growth and development. for conditional mutants an to essential to null mutants conditional mutants for and mutations are the used type of conditional mutations and are a in activity of the gene when the gene is a temperature. We the and of a Arabidopsis mutant affected in the PMM to restrictive temperature, pmm-12 mutant plants were PMM enzyme activity and protein and cell The pmm-12 mutant plants also had a lower AsA but is demonstrated that this AsA account for the cell death phenotype. we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype. Plant and thaliana plants were grown on Murashige and Skoog in at of The used was and the restrictive was 28 °C. The pmm-12 was for at °C. were of grown at and to in the of of were grown at for and for growth defects at restrictive temperature. grown on acid that showed growth defects within days after transfer to restrictive wild were mutants and to the on of the observed phenotype was plants of plants of were used to the the was and the M. D. A. Plant Sci. 2002; 7: Full Text Full Text PDF PubMed Scopus Google Scholar) plants were at the Plant J. 1998; PubMed Google Scholar). plants were and of of and a at °C. on were of The was of and were The was and in of and and pmm-12 mutants a pmm-12 mutants were back-crossed the wild type The plants were to and the was at restrictive temperature. mutant plants were transfer to and growth at the temperature. of mutant plants to was of the mutant was at plants and the of the mutant phenotype at restrictive temperature. mutant were wild In a a the G. P. J. K. M. T. 2004; PubMed Scopus Google Scholar, H. P. G. J. M. T. Plant Physiol. 2001; PubMed Scopus Google Scholar), was to plants to the of the PMM the from a of were as M. 2007; PubMed Scopus Google Scholar). were the of the analysis The a of were and mutants were for of the mutant In the of a mutants are the of mutant were for insertion/deletion of the G. Norris S.R. Last R.L. Plant Physiol. 2002; PubMed Scopus Google Scholar). In the of were of the the PMM gene no were were were on the Arabidopsis on the S. H. Mol. Biol. PubMed Google Scholar). The used for were as at of at at at °C a of °C of at at at at °C. were on the Arabidopsis on the S. H. Mol. Biol. PubMed Google Scholar) to and a of were amplified of and a of for The used for the were as at of at at on the at °C. were and to a the had been at °C for and were The for a were as at of at at at °C. were an of of PMM and its mutant were from wild type mutant plants as a The was the the from pmm-12 plants, and the to the wild type were and the of a at in of and were grown for at °C and of at temperature, of was proteins were as and as Protein were to PubMed Scopus Google Scholar). activity was a the of mannose 1-phosphate to mannose 6-phosphate was to the of to 6-phosphate the of isomerase and The was the in at as a of the in In PMM activity was as D. S. R. Plant Biol. Scopus Google Scholar). In in was in and Cell was and were on PMM activity was in a of protein a and The was the of the of and the at was for from at seedlings was In vitro activity of recombinant PMM was in the The PMM of recombinant protein was protein analysis and Protein AsA of and mutant Arabidopsis was as Chem. Scopus Google Scholar, C. J. D. PubMed Scopus (302) Google Scholar). concanavalin A of total protein from Arabidopsis was on a and a The was for in and for in to The was for were type and pmm-12 protein extracts for were as W. Last R.L. P.L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). Protein extracts were on an and a Arabidopsis PMM was recombinant protein as the The was used at a of and Arabidopsis protein-disulfide isomerase and were used at a of and was to a from at seedlings was of a Cell of Arabidopsis thaliana was for in growth and development when grown at a restrictive of 28 °C. mutants severe phenotypes were transfer to to to were to the of the in the of analysis of the mutants of impaired in the of plant and the of mutants that were in plant The observed phenotype was at restrictive temperature, in growth E. Scholar). of the in growth showed a growth cell death at restrictive temperature. plants to wild type plants, a analysis of the the of the not of the Mutant were of mutant plants wild type was for the that is and PubMed Scopus Google Scholar). were used for the In a of the of a total of plants, the mutant plants and as were an of G. P. J. K. M. T. 2004; PubMed Scopus Google Scholar). analysis of the of the mutant phenotype to a on the and the lower of In the in the and within the of were used for This the the mutant locus could to and the lower of Because analysis of not in the of for the an of plants was and within the This of the affected gene to a the and the lower and within the (Fig. showed to the mutant phenotype. of the mutant an of plants was the gene a and wild type plants could not on their phenotypes. to the of the wild type and the to their we only used the mutant for In mutants were and to from the G. Norris S.R. Last R.L. Plant Physiol. 2002; PubMed Scopus Google Scholar). and the were and used to the mutant were and in the the As a the could to and as the and the lower The were within the in a and that the gene (Fig. to the gene for in the Arabidopsis on the the the gene of the the was both in the mutant and wild type In mutant plants, we and in gene to the of the In the no were the of the the gene was in plants and in the mutant that had been and used the of Both mutations were from the wild type gene and in mutant not The Mutant a gene that the mutations a In the Arabidopsis only PMM gene is W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). we to the mutant as the pmm-12 mutant. The and both in the of wild type and mutant and their acid that in a to to a of a a The in a to an a (Fig. PMMs are to the of acid R.L. J. Mol. Biol. PubMed Scopus Google Scholar). of the a that is involved in the transfer V. M. G. Schaftingen E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Both mutations were this the affected a the involved the of an acid (Fig. and of a that the mutations in the locus were responsible for the pmm-12 pmm-12 plants were a the wild type of the a on of a also a when grown on at restrictive not This that the pmm-12 mutant phenotype could of the We the from the N. K. Plant Mol. Biol. PubMed Scopus Google Scholar). This an from the of the PMM as the on from seedlings N. K. Plant Mol. Biol. PubMed Scopus Google Scholar). of the plants grown from the that were to In no for could A at plants that the of plants to ∼25% of the of plants were from wild type not that a in causes The pmm-12 Mutant a Cell grown in vitro at temperature, pmm-12 mutant seedlings not phenotype when wild type when to 28 growth was and seedlings died within several days (Fig. The of cell death to the of at the of and in the (Fig. and after days at 28 °C. In plants grown in at °C a cell death only after to 28 °C for at (Fig. In mutant pmm-12 plants were and grown in at °C plants also growth and within after transfer to 28 °C (Fig. analysis of the of seedlings at temperature, the growth of pmm-12 was decreased that of wild type (Fig. transfer to restrictive temperature, days after growth of wild type plants significantly increased plants were to days In growth of pmm-12 seedlings was transfer to 28 °C. growth was arrested within days and could not the seedlings to °C after days at 28 °C (Fig. and were to and at 28 pmm-12 in to wild type could not not we demonstrated that the cell death phenotype is throughout both vegetative and reproductive development and that the of cell death on the pmm-12 PMM and Protein the of the pmm-12 on PMM activity in total PMM activity was in extracts from wild type and pmm-12 were grown for at °C and to 28 °C at °C. was and days after and PMM activity was a grown at pmm-12 plants had PMM activity significantly lower that of wild type the of the the in pmm-12 extracts was of that in wild type transfer to 28 wild type plants showed a in activity of activity in the mutant plants (Fig. a that pmm-12 plants had decreased PMM protein both at and 28 °C. Furthermore, the to 28 °C had a on protein levels in wild type and in mutant plants (Fig. We that pmm-12 plants from a in both total PMM activity and PMM protein levels. The Mutant PMM the of of the mutations on recombinant enzyme different Arabidopsis PMM were the wild type of the mutations and and both mutations of the proteins were at both and 28 °C. The different PMM the of wild type PMM and were of both and were of and to wild type The of wild type PMM was 28 that of the of was the mutations in a catalytic efficiency that was only of the wild type of on were observed 1). that the in catalytic activity of the mutant enzyme is due to the the in catalytic efficiency of observed at 28 °C is that at of recombinant wild type and mutant Arabidopsis PMM at and 28 °C were in a The are in a The pmm-12 Cell AsA of PMM levels gene in N. has been to in decreased AsA levels W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). we AsA levels in pmm-12 after transfer to restrictive temperature, the vtc1 mutant (11Conklin P.L. Norris S.R. Wheeler G.L. Williams E.H. Smirnoff N. Last R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4198-4203Crossref PubMed Scopus (302) Google Scholar, 12Conklin P.L. Pallanca J.E. Last R.L. Smirnoff N. Plant Physiol. 1997; 115: 1277-1285Crossref PubMed Scopus (166) Google Scholar) as a In wild type plants, AsA levels increased significantly after transfer to 28 in pmm-12 plants and to only of wild type levels. In fact, AsA in pmm-12 seedlings was that in when (Fig. of not their AsA vtc1 seedlings showed growth cell death transfer to 28 °C. In we to the phenotype of pmm-12 the of AsA levels increased in pmm-12 seedlings grown on cell death could not after transfer to 28 °C not that the cell death phenotype observed in pmm-12 plants is not AsA Protein in the pmm-12 Mutant is is required for the synthesis of the core glycan chain that is attached to R.G. Glycobiology. 2002; 12: 43R-56RCrossref PubMed Scopus (999) Google Scholar). we the of the on total a that to the mannose of L. 1985; PubMed Scopus Google Scholar). in protein were observed in extracts from pmm-12 mutant plants to 28 °C extracts from plants at °C wild type was a but in the of at to a lower of were transfer to 28 °C (Fig. A in has been to in the of to a that as a on a protein W. Last R.L. P.L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, G. C. M. C. A. H. P. Plant J. PubMed Scopus Google Scholar). is an protein of the that is modified J. Plant Physiol. PubMed Scopus Google Scholar, H. G. G. 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The was to a on the lower of a of in in the were of to from plants on of the This the of from the in a of the of that the affected gene a PMM that were mutations in the of the gene from the pmm-12 mutant the wild type gene in the The pmm-12 Mutant a for of Plant on the Arabidopsis a PMM gene the of this was that the protein showed PMM mannose 1-phosphate to mannose 6-phosphate W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). and gene in Arabidopsis and N. a role for PMM in AsA no mutants were and to were The lack of such mutants impaired of the involvement of PMM in AsA biosynthesis W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). the conditional phenotype of the pmm-12 mutant has to PMM in The that the pmm-12 mutant is a to the of the Arabidopsis PMM gene and to the role of PMM and GDP-mannose in plant growth and development. PMM the and are of the of acid that catalyze transfer R.L. J. Mol. Biol. PubMed Scopus Google Scholar). of the acid a of a core and a the at the of the is a in the core V. M. G. Schaftingen E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) that has a the that is in and length Zhang W. Zhang G. D. PubMed Scopus Google Scholar, Zhang C. J. D. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Both the and mutations are in the core the J. Mol. Biol. 1999; PubMed Scopus Google Scholar), the Jones PubMed Scopus Google Scholar), and the of as a Zhang C. J. D. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), we that the affected acid are not essential for not the mutations the of the core because both acid are The a a in the of an a In the affected is PMMs in not that is for of the In vitro the different recombinant mutant PMM at both and 28 showed that the in a in PMM enzyme the only affected enzyme 1). This due to the of the acid the have The activity also that the observed in catalytic efficiency of is of the temperature. the observed in PMM protein and enzyme activity observed at °C (Fig. that the pmm-12 mutant is not a mutant in the that the protein only at restrictive temperature. the that Arabidopsis plants at °C PMM in plants at 28 °C this Protein the Cell PMM enzyme activity lead to GDP-mannose deficiency and have pleiotropic PMM has been to an role in the biosynthesis of AsA (2Wheeler G.L. Jones M.A. Smirnoff N. Nature. 1998; 393: 365-369Crossref PubMed Scopus (795) Google Scholar, 3Qian W. Yu C. Qin H. Liu X. Zhang A. Johansen I.E. Wang D. Plant J. 2007; 49: 399-413Crossref PubMed Scopus (82) Google Scholar). we pmm-12 AsA wild type after transfer to restrictive temperature, AsA levels in the pmm-12 mutant were only of in wild type plants and in the AsA mutant vtc1 (Fig. This the for PMM involvement in AsA this was that not cell death when to 28 °C. the pmm-12 mutant the of not the of cell death after transfer to 28 °C not that the cell death phenotype observed in the mutant is not AsA its role in AsA GDP-mannose also a key role in modifications, such as protein is essential for and of The core glycan attached to is on a and mannose are to from GDP-mannose (4Lerouge P. Cabanes-Macheteau M. Rayon C. Fischette-Lainé A-C. Gomord V. Faye L. Plant Mol. Biol. 1998; 38: 31-48Crossref PubMed Google Scholar). The after the a deficiency in in pmm-12 In a post-translational protein on GDP-mannose T. N. Curr. Opin. Chem. Biol. 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In mutant of the gene a protein for complex and the genes and different of the Arabidopsis have been to in to G. C. M. C. A. H. P. Plant J. PubMed Scopus Google Scholar, M. Gomord V. C. N. F. P. Faye L. M. L. J. 2001; PubMed Scopus Google Scholar, P. J. C. J. Cell Biol. 2002; PubMed Scopus Google Scholar, H. F. N. J. A. Plant Cell. PubMed Scopus Google Scholar). The Arabidopsis gene and in lethality embryonic development at a early for the lethality of a severe of the phenotype an of N-glycosylation. This to the that a deficiency in this account for of the of W. Last R.L. P.L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). is to that acts downstream of PMM in the GDP-mannose biosynthesis (Fig. 1). similar to the pmm-12 could not AsA W. Last R.L. P.L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), that in both the lack of AsA is not to the and cell The mutant is to a mutant in its AsA biosynthesis P.L. Pallanca J.E. Last R.L. Smirnoff N. 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Plant J. 2007; PubMed Scopus Google Scholar). that pmm-12 the GDP-mannose AsA biosynthesis at the of of the of AsA synthesis in the mutants and pmm-12 are required in to of in this is the of the is to that the of such as of mannose 6-phosphate a role for mannose 1-phosphate in cell We and for and for and for the We also R. for the and C. for the