Vacuolar Processing Enzymes Are Essential for Proper Processing of Seed Storage Proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the βVPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: αVPE, βVPE, and/or γVPE. More than 90% of VPE activity is abolished in the βvpe-3 seeds, and no VPE activity is detected in the αvpe-1/βvpe-3/γvpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to βvpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (αvpe-1 and γvpe-1) and double (αvpe-1/γvpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, αVPE and γVPE, are not necessary for precursor processing in the presence of βVPE, but partly compensates for the deficiency in βVPE in βvpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type βVPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement βVPE activity in this processing. The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the βVPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: αVPE, βVPE, and/or γVPE. More than 90% of VPE activity is abolished in the βvpe-3 seeds, and no VPE activity is detected in the αvpe-1/βvpe-3/γvpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to βvpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (αvpe-1 and γvpe-1) and double (αvpe-1/γvpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, αVPE and γVPE, are not necessary for precursor processing in the presence of βVPE, but partly compensates for the deficiency in βVPE in βvpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type βVPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement βVPE activity in this processing. In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) 1The abbreviations used are: PSV, protein storage vacuole; ATPF, amino-terminal processed fragment; IPF, internal processed fragment; AP, aspartic proteinase; VPE, vacuolar processing enzyme; WT, wild-type; MCA, 4-metyl-coumaryl-7-amide. as a source of nitrogen for growth after seed germination (1Muntz K. Plant Mol. Biol. 1998; 38: 77-99Crossref PubMed Google Scholar). During seed maturation, storage proteins are synthesized on the rough endoplasmic reticulum as proprotein precursors and are then transported into PSVs by a vesicle-mediated pathway (2Robinson D.G. Baumer M. J. Plant Physiol. 1998; 152: 659-667Crossref Scopus (57) Google Scholar). Upon arrival at the vacuoles, the proproteins are converted into their respective mature forms by limited proteolysis at specific sites. The conversion of each storage protein is known to occur at the C-terminal side of an asparagine residue in the proprotein, suggesting a single type of proteinase that has substrate specificity toward an asparagine residue is responsible for the processing of storage protein precursors (3Hara-Nishimura I. Shimada T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1995; 145: 632-640Crossref Scopus (104) Google Scholar, 4Hara-Nishimura I. Maeshima M. Robinson A.D.G. Rogers J.C. Vacuolar Compartments in Plants. Sheffield Academic Press, London, UK2000: 20-42Google Scholar). Previously (5Hara-Nishimura I. Inoue K. Nishimura M. FEBS Lett. 1991; 294: 89-93Crossref PubMed Scopus (218) Google Scholar, 6Hara-Nishimura I. Takeuchi Y. Nishimura M. Plant Cell. 1993; 5: 1651-1659Crossref PubMed Scopus (135) Google Scholar), we identified an enzyme responsible for the maturation of various storage proteins and designated it vacuolar processing enzyme (VPE). In vitro processing assays using purified VPE and seed protein precursors showed that VPE is capable of processing several seed proteins, including the 2 S albumins and 11 S globulins, of the pumpkin and castor bean (5Hara-Nishimura I. Inoue K. Nishimura M. FEBS Lett. 1991; 294: 89-93Crossref PubMed Scopus (218) Google Scholar, 7Shimada T. Hiraiwa N. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1994; 35: 713-718Crossref PubMed Scopus (73) Google Scholar). VPEs exhibit substrate specificity toward an asparagine residue, the amino acid well conserved at the P1 position in the processing sites of storage protein precursors in many plant species. Therefore, we proposed that the VPE-mediated processing system plays a central role in the maturation of various seed proteins in PSVs (3Hara-Nishimura I. Shimada T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1995; 145: 632-640Crossref Scopus (104) Google Scholar, 4Hara-Nishimura I. Maeshima M. Robinson A.D.G. Rogers J.C. Vacuolar Compartments in Plants. Sheffield Academic Press, London, UK2000: 20-42Google Scholar). VPEs belong to a novel family of cysteine proteinases (C13; EC 3.4.22.34) (8Hara-Nishimura I. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London, UK1998: 746-749Google Scholar). VPE homologs are found in various organisms, including plants (4Hara-Nishimura I. Maeshima M. Robinson A.D.G. Rogers J.C. Vacuolar Compartments in Plants. Sheffield Academic Press, London, UK2000: 20-42Google Scholar, 9Hara-Nishimura I. Kinoshita T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1998; 152: 668-674Crossref Scopus (65) Google Scholar), mice (10Chen J.M. Dando P.M. Stevens R.A. Fortunato M. Barrett A.J. Biochem. J. 1998; 338: 111-117Crossref Scopus (114) Google Scholar, 11Shirahama-Noda K. Yamamoto A. Sugihara K. Hashimoto N. Asano M. Nishimura M. Hara-Nishimura I. J. Biol. Chem. 2003; 278: 33194-33199Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), humans (12Halfon S. Patel S. Vega F. Zurawski S. Zurawski G. FEBS Lett. 1998; 438: 114-118Crossref PubMed Scopus (51) Google Scholar), and the protozoan, Schistosoma mansoni (13Klinkert M.-Q. Felleisen R. Link G. Ruppel A. Beck E. Mol. Biochem. Parasitol. 1989; 33: 113-122Crossref PubMed Scopus (157) Google Scholar). Plant VPE homologs are separated into two subfamilies: one specific to seeds and the other specific to vegetative organs (9Hara-Nishimura I. Kinoshita T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1998; 152: 668-674Crossref Scopus (65) Google Scholar, 14Muntz K. Shutovb A.D. Trends Plant Sci. 2002; 7: 340-344Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We have identified three VPE genes in Arabidopsis, αVPE, βVPE, and γVPE (15Kinoshita T. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1995; 36: 1555-1562PubMed Google Scholar, 16Kinoshita T. Nishimura M. Hara-Nishimura I. Plant Mol. Biol. 1995; 29: 81-89Crossref PubMed Scopus (58) Google Scholar). Northern blot analysis and histochemical analysis with β-glucuronidase reporter constructs showed that αVPE and γVPE are expressed in vegetative organs, whereas βVPE is expressed in seeds (17Kinoshita T. Yamada K. Hiraiwa N. Nishimura M. Hara-Nishimura I. Plant J. 1999; 19: 43-53Crossref PubMed Scopus (189) Google Scholar). Recently, a fourth VPE, designated δVPE, was identified and shown to be expressed in maturing seed at an early stage (18Gruis D.F. Selinger D.A. Curran J.M. Jung R. Plant Cell. 2002; 14: 2863-2882Crossref PubMed Scopus (106) Google Scholar). They reported Arabidopsis mutants in which the βVPE or δVPE gene was disrupted by the insertion of a transposon. Surprisingly, most 12 S globulin and 2 S albumin proteins are processed normally in βvpe seeds, and only a small proportion of the storage proteins remain as unprocessed or partially processed forms. In the δvpe seeds, no detectable change was observed in the processing of storage proteins. On the basis of (18Gruis D.F. Selinger D.A. Curran J.M. Jung R. Plant Cell. 2002; 14: 2863-2882Crossref PubMed Scopus (106) Google that other proteinases are in the processing of storage proteins in Arabidopsis seeds two of storage proteins, 12 S globulins to as and 2 S albumins are cleaved at an to produce and which are by a Plant Mol. Biol. PubMed Scopus Google Scholar). This processing is conserved known 12 S The processing of the 2 S albumins is than that of the 12 S 2 S albumins are of two small and which are by E. A. J. J. M. G. M. J. Plant Physiol. PubMed Google Scholar). During the maturation of the 2 S albumins, the pro2S albumins are cleaved at no than in the of three the processed the internal processed and the C-terminal processed The for processing storage proteins have with in to no has to in the processing of seed storage proteins in We a analysis of the genes responsible for this processing in mutants that accumulate storage protein precursors isolated by All six mutants a defect in the βVPE that βVPE is in the processing of storage proteins. we found that the vegetative-type VPEs, αVPE and γVPE, in this with Plant and and and used as of Arabidopsis and then on or that and and at of Arabidopsis of 12 S and 2 S Arabidopsis of three different that we with several different The other two K. and T. seeds the of to and then to with S globulin and S albumin to the storage protein We isolated the six mutants the was isolated βvpe-3 and isolated and isolated of the of βVPE was into and with the a precursor of 2 S albumin of Arabidopsis was into and E. with the The proteins purified with a The purified proteins used for of specific βVPE or 2 S albumin by the T. E. K. Y. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 2002; PubMed Scopus (73) Google Scholar). of the purified proteins in of was with an of seeds of Arabidopsis to and The protein to and of 12 S globulin the and with was into a two with at after the was and the was The βVPE purified using a and seeds of or mutants in a and The at for and then at for The to by to a in a The with in and and then with each of 2 S albumin 12 S globulin and βVPE in the for used as detected with an and with that separated on the with to on a In the of the protein found in seeds of the blot was with in and with the blot was to the of of βVPE isolated of and mutant to the βVPE gene by using a and of with a using specific for βVPE gene and of αVPE βVPE and γVPE by isolated insertion mutants of each VPE a large of Arabidopsis plants by and by the of A. we used the for and for mutants of αVPE (αvpe-1 and and two mutants of βVPE and a insertion at the a insertion at the and a insertion at the of each gene. the of A. of we used the for and for mutant of γVPE was a insertion at the To the we used specific and of and of double and triple mutants of VPEs, we the VPE mutants as plant of was with a plant of We isolated a plant of double mutant by The plant of double mutant was with a plant of plants of double mutant and αvpe-1/βvpe-3/γvpe-1 triple mutant isolated or mutants a of and To a double we a plant of with a plant of plant of double mutant was isolated This double mutant a To the βvpe-3 we used the was by with the and the and The was with The βvpe-3 was to whereas the βVPE was of VPE activity was as M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 2002; PubMed Scopus Google Scholar). We used a The enzyme seeds was with in an and The was using The was an of and an of of by synthesized two for and for and used as the the of 2 S The of each and the seeds with or in of and The to for at and the of the to at and in was in of at seeds and the mutant plants for with a that of and in The then into of than in with a and to and by the I. Takeuchi Y. Nishimura M. Plant Cell. 1993; 5: 1651-1659Crossref PubMed Scopus (135) Google Scholar). on a and with and with a 2 by a of by a in the βVPE in Arabidopsis of Arabidopsis to mutants with a defect in the maturation of seed storage proteins. seeds of to analysis with a storage 2 S albumin or 12 S We six mutant that forms of storage proteins in the seeds. Previously (3Hara-Nishimura I. Shimada T. Hiraiwa N. Nishimura M. J. Plant Physiol. 1995; 145: 632-640Crossref Scopus (104) Google Scholar), we reported that storage protein precursors are converted into the respective mature forms by the of seed-type VPE. Therefore, the of forms of storage proteins be by a defect in the maturation as in To βVPE is in the mutant seeds, we used analysis with βVPE of and detected on a blot of proteins the seeds of and In of was detected in the seeds of isolated that functional βVPE is lacking in mutants mutants have a in the βVPE as shown in showed that six mutants have a defect at the Therefore, designated to the in the the and the of the βVPE gene. has a for to insertion in the a in that no functional βVPE is in or with the of The two βvpe-3 and have the which an amino acid for to for βvpe-3 and the two βVPE of and on in βVPE is conserved in the VPE suggesting that this residue is for the we isolated two and has a insertion in the and has an insertion in the They to produce the two βVPE on not of in βvpe-3 a of seed storage protein of and βvpe-3 seeds on an with The seeds the and of the 12 S globulins and the large and small of the 2 S albumins In the βvpe-3 seeds and proteins in to the mature proteins The seeds of the other βvpe mutants protein not and with a of the forms than and with The amino acid of the proteins in the βvpe-3 seeds as shown in The of the and proteins that the 2 S albumins, and proprotein precursor of 2 S pro2S is of an ATPF, a small by an IPF, and a large of the proteins was to be an processed precursor of at the and which is with the substrate specificity of VPE The protein was a precursor of after the of the by cleavage of the and which is with the substrate specificity of VPE protein was a precursor of after the of the by cleavage of the and results suggest that the βvpe-3 seeds accumulate several 2 S albumin precursor forms with no with the substrate specificity as βVPE be in the of the The of the and proteins are with the the cleavage sites of the of the 12 S globulins, and On the basis of their proteins are the proprotein precursors of the 12 S This that the processing of the precursors of 12 S globulins is abolished in the βvpe-3 seeds. In contrast to the of precursor forms of the 2 S albumins and 12 S globulins, large amounts of storage proteins as the mature forms in the βvpe-3 seeds. This the of the mature forms of the 12 S globulins by processing at the and in the βvpe-3 seeds. To we the of the and of the 12 S globulins of and βvpe-3 seeds. The of the of the βvpe-3 12 S globulins to of the normally processed of the 12 S globulins results suggest that with the substrate specificity as βVPE is in the processing of storage protein VPEs, αVPE and γVPE, in the of results that vegetative VPEs are in the processing of storage protein precursors with seed-type To investigate this we isolated the and mutants a large of of The mutant has a single insertion in the of the αVPE gene on 2 and The mutant has in the of the γVPE gene on and To the of on the of the αVPE and γVPE we the mutant plants and to and Northern blot we showed that the αVPE and γVPE genes are in Therefore, we used for plants αVPE and γVPE in On the other no αVPE and no γVPE and results that both and are mutants that are to produce the respective We further generated double and triple mutants on the single and To the deficiency in functional VPE we VPE activity in mutant seeds, using a that the VPE activity detected was in seeds of the single mutant βvpe-3 and in the two double and VPE activity was not detected at in seeds of the triple results that both αVPE and γVPE are responsible for a small of the VPE activity in seeds, whereas βVPE is responsible for a of VPE To the of αVPE and γVPE are in processing storage protein we the storage proteins that accumulate in the seeds of each mutant. that no detectable 12 S globulin or 2 S albumin precursors in the seeds of the single and or of the double mutant This that both αVPE and γVPE are in the presence of the double mutant storage protein precursors and mature forms than the single βvpe-3 suggest that γVPE activity compensates for the of βVPE activity in the βvpe-3 seeds, is no for γVPE in the presence of mature forms of the storage proteins detected in the seeds of the triple αvpe-1/βvpe-3/γvpe-1 mature forms of storage proteins in the double This that αVPE to the processing of storage protein The analysis of various mutants lacking the of the vegetative-type VPEs, αVPE and γVPE, in the processing of storage protein precursors with the seed-type VPE, The protein of the triple mutant seeds was different that of seeds. We the of the PSVs of the mutant seeds. of the and and triple mutant seeds. found the and mutant seeds, and the PSVs normally in the and of mutant results that the precursor forms of storage proteins accumulate in the PSVs of the mutant seeds. of Proteolytic of in the of have shown that storage protein precursors are processed in the absence of functional The mechanism is in this To proteins in the seeds of the triple to and a The was with and into each of which was to We not the mature 12 S globulins that are by cleavage of the at the cleavage most 12 S proteins as proprotein we detected and the 12 S and to by cleavage of the or the cleavage The of and are with the the of They to by cleavage the cleavage that most of the This VPEs are are processed at sites the VPE processing In contrast to the of pro2S albumins in the seeds of the triple mutant. We detected 2 S albumin with various analysis of proteins showed that the proteins cleaved at the or the in the and the in the that a deficiency in VPE activity alternatively processed 2 S Therefore, proteinase the or the be in the seeds. be that which the conserved was converted into the mature protein by cleavage of the in the in seeds VPEs have no to Therefore, proteinase responsible for the processing of pro2S albumins in the absence of VPEs in the processing of the of in 2 S then of proteinase is in the processing of and in the processing of the other 2 S albumins in the absence of it was reported that an purified seeds of various plants cleaved the of a with a of K. J. M. J. E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, N. M. Nishimura M. Hara-Nishimura I. J. Biochem. PubMed Scopus Google Scholar). To Arabidopsis seeds has the to the of 2 S albumins, we synthesized with to the of and and used as for a Arabidopsis seeds. the was to that the substrate by in with the of three by The was by the of an A. the generated that cleaved the and the of the and the of the results that Arabidopsis the of and by VPE by cleaved by is conserved in the of the pro2S albumins that AP, with VPE, is in the processing of the pro2S This that for the deficiency in VPEs in the the forms of the 2 S albumins, and accumulate in the seeds of the βvpe mutant and the triple mutant αvpe-1/βvpe-3/γvpe-1 The processing of pro2S albumins was partially abolished in the absence of This that VPE activity is for the cleavage of the of the 2 S The initial processing of the pro2S albumins by VPE their cleavage by The proprotein precursor the at the that is conserved in the other 2 S albumins but the at the conserved This that the processing of the precursor in a the of was detected in the absence of βVPE 2 and This that βVPE plays role in the conversion of the precursor into the respective To the of βVPE, we the processing of the precursor seed We the of the proteins that in the maturing seeds of and βvpe-3 plants at different to the cleavage sites the of the precursor In the maturing seeds, the precursor was cleaved at the and then cleaved at the On the other in the maturing βvpe-3 seeds, the precursor was cleaved at the but not at the results that βVPE is in the initial cleavage at the This is with that Arabidopsis VPEs have the to the on the C-terminal side of not only but of the initial cleavage the cleavage by with the of was not detected in the early of maturation in βvpe-3 seeds. The of cleavage was in the absence of Our results that VPEs are in the processing of storage proteins in maturing Arabidopsis seeds. Previously (5Hara-Nishimura I. Inoue K. Nishimura M. FEBS Lett. 1991; 294: 89-93Crossref PubMed Scopus (218) Google Scholar, 7Shimada T. Hiraiwa N. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1994; 35: 713-718Crossref PubMed Scopus (73) Google Scholar), we showed that the purified VPE the castor bean the proprotein precursors of the storage proteins, 11 S globulin and 2 S albumins, to produce the respective mature forms. results in and in vitro that VPEs as processing in the maturation of storage proteins. In Arabidopsis, three βVPE, and in the processing of storage proteins. the βVPE is the enzyme most essential for this processing. This is in with the that the isolated mutants that accumulate storage protein precursors in their seeds have a defect in the βVPE gene. Previously (17Kinoshita T. Yamada K. Hiraiwa N. Nishimura M. Hara-Nishimura I. Plant J. 1999; 19: 43-53Crossref PubMed Scopus (189) Google Scholar), we showed that αVPE and γVPE are in the vacuoles of vegetative and various vegetative-type VPEs found to in maturing seeds. G. Robinson D.G. J. Cell 1995; Google reported that seeds have vegetative vacuoles, which are by PSVs seed This that the vegetative-type VPEs remain in the PSVs of maturing seeds. seeds a of vegetative VPE activity in contrast to the of βVPE vegetative-type VPEs for the in VPE activity in βvpe seeds, not in the presence of fourth Arabidopsis VPE, δVPE, was shown to be expressed in the maturing seeds at an early stage (18Gruis D.F. Selinger D.A. Curran J.M. Jung R. Plant Cell. 2002; 14: 2863-2882Crossref PubMed Scopus (106) Google Scholar). the processing of storage proteins is abolished in the triple δVPE to no role in this processing. is that δVPE is expressed in other than that accumulate storage proteins, or is at other than the VPEs the of a to produce an and a which are by a The VPE-mediated processing activity is that storage protein precursors are detected in maturing seeds. In in the mutant αvpe-1/βvpe-3/γvpe-1 seeds, mature 12 S globulins Instead, a small proportion of 12 S globulin molecules are cleaved at the or the to the processing to produce two The cleavage activity was that most 12 S globulin molecules as precursor forms in the mutant seeds. cleaved forms not detected in the seeds. The cleavage in the mutant seeds that the the processing sites are and to by not only VPEs but other VPEs the of storage 2 S albumin to produce a small and a large by a E. A. J. J. M. G. M. J. Plant Physiol. PubMed Google Scholar). in mutant αvpe-1/βvpe-3/γvpe-1 seeds, a proportion of the 2 S albumin molecules are cleaved at the in the to produce two by the of most of the pro2S albumin molecules accumulate as precursors in the mutant seeds, and forms are not detected in the maturing seeds. results that activity is not to the 2 S albumin precursor be responsible for the to the than for the precursors into two the for the processing of 2 S albumins in The amino acid the and of the 2 S albumins are We found that each has a processing the VPE and the Our results suggest that the VPE in the processing of the pro2S albumins to produce the two and the in the is that VPE cleavage a change in the 2 S albumins to to cleavage by This is in with the that mutants isolated in this have a defect in the βVPE but not in the gene. we reported that VPE and seed maturation and after seed germination at both the protein and activity N. M. Nishimura M. Hara-Nishimura I. J. Biochem. PubMed Scopus Google Scholar, N. Takeuchi Y. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 1993; Scholar). This that two in the processing of storage proteins. The 2 S is in no to be cleaved by VPE. the precursor is to processing seed The in the is cleaved by VPE to produce two and then the three amino of the large are by cleavage of the by VPE was to be an we found that Arabidopsis VPE at the C-terminal side of aspartic acid M. Nishimura M. Hara-Nishimura I. Plant Cell Physiol. 2002; PubMed Scopus Google Scholar). VPE a different in than in the other 2 S albumins, the processing by VPE and is to that observed in the other 2 S in the absence of both the 2 S albumins and 12 S globulins to accumulate normally in seeds. The of PSVs in the VPE triple mutant seeds is that in the seeds. The mutant seeds normally and During the forms of storage proteins are in the mutant seeds not The role of the processing of seed storage proteins to be We of for for of specific Arabidopsis 2 S We Y. for on