2010.07.14 二硫键异构酶在水稻内质网成熟中的作用[PCP]

2 Running title: Role of PDIL1-1 in proglutelin maturation in rice *Correspondence author Name: Toshihiro Kumamaru Address: Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan Telephone / Fax number: 81-92-642-3057 e-mail: kumamaru@agr.kyushu-u.ac.jp Subject Area: Proteins, enzymes and metabolism Structure and function of cells Number of black and white figures: 5 Number of color figures: 2 Number of tables: 2 © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org Plant and Cell Physiology Advance Access published July 13, 2010 at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 4 Protein Disulfide Isomerase Like 1-1 Participates In The Maturation Of Proglutelin Within Endoplasmic Reticulum In Rice Endosperm Mio Satoh-Cruz1, 2, †, Andrew J. Crofts2, 5,†, Yoko Takemoto-Kuno1, 4, †, Aya Sugino1, 2, Haruhiko Washida2, 6, Naoko Crofts2, 5, Thomas W. Okita2, Masahiro Ogawa3, Hikaru Satoh1 and Toshihiro Kumamaru1, * 1 Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan 2 Institute of Biological Chemistry, Washington State University, Pullman WA, 99164-6340 USA 3 Faculty of Human Life Science, Yamaguchi Prefectural University, Sakurabatake 3-2-1, Yamaguchi 753-8502, Japan Present address: 4 Present address: National Institute of Crop Science, Kannondai 2-1-18, Tsukuba 305-8518, Japan. 5 Present address: International Liberal Arts Program, Akita International University, Akita, 010-1292, Japan 6 Present address: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 5 Footnote: † These authors contributed equally to this work. Abstract The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal-ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its buildup in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as for the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, their deposition within prolamine protein bodies in esp2 suggests that PDI1-1 helps retain proglutelin in the cisternal ER lumen until it is attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides. Keywords: endoplasmic reticulum, endosperm, Oryza sativa, protein body, protein disulfide isomerase, storage protein at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 6 Introduction Rice seed storage proteins consist mainly of two classes (Juliano 1972). One class consists of the acid-soluble glutelins, which are homologous to the 11S globulins of soybean and pea (Shotwell and Larkins 1989, Takaiwa et al. 1987, Zhao et al. 1983). The other class is the alcohol soluble prolamines, the storage protein class typically found in cereals (Ogawa et al. 1987, Shewry and Tatham 1999). Rice seed also accumulates a salt-soluble globulin which comprises of up to 5% of the total seed protein (Padhye and Salunkhe 1979). Glutelins are initially synthesized as a 57 kD precursor on the endoplasmic reticulum (ER) (Yamagata et al. 1982). The precursor is then exported to the protein storage vacuole (PSV; also called protein body II, PB-II) where it is post-translationally processed into acidic and basic subunits interlinked by a disulfide chain (Krishnan and Okita 1986, Yamagata et al. 1982). The glutelin-containing PSV is characterized by its irregular shape with a diameter of about 3-4 µm and high uniform staining density (Beachtel and Juliano 1980, Tanaka et al. 1980). The prolamines are also synthesized on the ER membrane but, unlike proglutelins, are retained in the ER lumen to form spherical intracisternal inclusions 1-2 μm in diameter called PB-I (Beachtel and Juliano 1980, Ogawa et al. 1987, Tanaka et al. 1980). Prolamines lack an ER retrieval signal and, hence, their retention within the ER lumen and at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 7 assembly into an intracisternal inclusion granule is due to other mechanisms. One process that facilitates prolamine ER retention is RNA sorting (Hamada et al. 2003, Li et al. 1993) whereby prolamine RNAs are localized specifically to the ER (PB-ER) membranes that delimit PB-I. The enrichment of prolamine RNAs on the PB-ER would effectively concentrate the newly synthesized polypeptides within a confined ER lumenal space, favoring protein-protein interactions and assembly to form an intracisternal inclusion granule (Okita and Rogers 1996). In contrast, glutelin RNAs are enriched on adjacent cisternal ER membranes which together with PB-ER constitute the cortical ER complex in developing rice endosperm cells (Hamada et al. 2003, Li et al. 1993). A second process that facilitates the ER retention and assembly of prolamine polypeptides is the specific involvement of binding protein (BiP) (Muench et al. 1997). Although this lumenal chaperone is an excellent marker for ER, it is asymmetrically distributed within this membrane complex in rice endosperm cells (Li et al. 1993, Muench et al. 1997). BiP is highly enriched at the periphery of PB-I compared to the rest of the cortical ER (Li et al. 1993, Muench et al. 1997). Available evidence suggests that this lumenal chaperone facilitates the transport of the nascent prolamine polypeptide across the ER membrane and their folding and assemble into an intracisternal inclusion granule (Li et al. 1993, Muench and Okita 1997, Muench et al. 1997, Okita et al. 1998, Okita and Rogers 1996). Other lumenal chaperones such as protein disulfide isomerase (PDI) are also likely to at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 8 be involved in storage protein folding and intracellular transport. PDI, a catalyst of disulfide-bond formation and rearrangement (Rowling and Freedman 1993), is also a molecular chaperone which facilitates polypeptide folding and has been suggested to have a role in storage protein biogenesis. (Bulleid and Freedman 1988). The rice esp2 mutation was identified by the accumulation of abnormally large quantities of proglutelin with corresponding reductions in mature glutelin subunits (Kumamaru et al. 1987, Kumamaru et al. 1988). The esp2 endosperm was also devoid of an endosperm-specific PDI (Accession no. AB039278, PDIL1-1), an observation suggesting a role for this lumenal chaperone in the folding and maturation of proglutelin to a conformation competent for ER export. In the absence of PDIL1-1, proglutelin and prolamines co-assembled via intermolecular disulfide bonds to form numerous small intracisternal aggregates within ER (Takemoto et al. 2002). Although the available evidence indicates that PDIL1-1 is involved in glutelin trafficking, the exact role of this molecular chaperone in this process is not known. In this study, we show conclusively that the Esp2 locus is the structural gene for the PDIL1-1 and that the deficiency of this lumenal chaperone mediates the abnormal accumulation of proglutelin during rice endosperm development. The dependence on PDIL1-1 for the ER export of proglutelin is conditional and is influenced by temporal gene expression patterns of both glutelin and prolamine. As proglutelin and prolamine are normally restricted to distinct lumenal compartments due to the localization of their RNAs to specific ER subdomains, the abnormal interaction between these storage proteins at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 9 suggests that PDIL1-1 has an another role in addition to its disulfide isomerase and chaperone activities in facilitating the maturation of proglutelin to a state competent for ER export. Results Abnormal accumulation of proglutelin in the esp2 mutant is caused by the deficiency of PDIL1-1 To confirm that esp2 is a defective PDIL1-1 gene, we initiated several genetic studies. Gene dosage effect studies were carried out by generating F1 seeds obtained from reciprocal crosses between an esp2 mutant line “CM1787” and the wild type “Kinmaze” (Fig. 1). Densitometric measurement of PDIL1-1 protein obtained by immunoblot analysis showed that the amount of PDIL1-1 protein in the duplex (++e), simplex (+ee), nulliplex (eee) genotypes was 64%, 36% and 0%, respectively, of the wild type condition (Fig. 1B, C). Thus, the level of the PDIL1-1 protein increased linearly with the number of dominant Esp2 alleles, indicating that the amount of PDIL1-1 protein corresponded to the gene dosage of Esp2 allele. By contrast, the amount of proglutelin decreased according to the increase in the number of dominant Esp2 alleles. Relative to the amount of proglutelin detected in nulliplex genotype, the amounts were 24% and 8% in the simplex and duplex genotypes, respectively, corresponding to an inverse relationship between proglutelin levels at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 10 and number of Esp2 alleles. The level of mature glutelin subunits followed the increase in dominant Esp2 alleles, i.e. 34% and 64% in the simplex and the duplex, respectively, in comparison to the triplex. In contrast to the increase in mature glutelin subunit levels, the extent of the decrease in proglutelin levels was not linear with the increase in number of the Esp2 alleles. To obtain further genetic evidence to show that elevated proglutelin levels were caused by deficiency in PDIL1-1, we analyzed 206 F2 seeds derived from a cross between wild type Kinmaze and the esp2 mutant EM44. The level of PDIL1-1 protein in all 154 F2 seeds showing normal levels of proglutelin was the same as that detected in wild type (Table 1). On the other hand, PDIL1-1 was absent in the 52 F2 seeds showing elevated levels of proglutelin. These results indicate that the abnormal accumulation of proglutelins in esp2 mutants is due to the lack of PDIL1-1 protein. To determine whether the esp2 mutation was due to a lesion in the structural gene for PDIL1-1 itself or a gene regulating the transcription or modifying the expression of the gene coding the PDIL1-1, RFLP studies were conducted with progenies derived from a cross between indica rice cultivar “Kasalath” and the esp2 mutant, CM1787, using the PDIL1-1 clone (AB039278) as a probe. RFLP analysis of the F2 population showed that all esp2 homo genotypes co-segregated completely with a RFLP for the PDIL1-1 of CM1787 (Table 2), suggesting that esp2 is a mutation in the structural gene for PDIL1-1. at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 11 The esp2 gene encodes PDIL1-1 A full length PDIL1-1 cDNA clone was isolated from a cDNA library by using the partial cDNA clone (Accession No. AB039278) as a probe. The full-length cDNA clone (Accession No. AB373950) was 1,903 bp in length and contained a single open reading frame of 1,536 bp coding for 512 amino acids (Supplementary Fig. S1). PDIL1-1 contained a C-terminal ER retrieval tetra peptide KDEL, a potential glycosylation site and two thioredoxin active sites CXXC. The rice PDIL1-1 primary sequence showed 84.8%, 84.2% and 83.9% sequence identity to that of maize (Li and Larkins 1996), barley (Chen and Hayes 1994) and wheat (Shimoni et al. 1995) sequences, respectively. The corresponding gene sequence was identified on the BAC clone OSJNBa0058P12 (AC139170) of chromosome 11 by “blast” searching the Rice Genome Automated Annotation system (RiceGAAS, http://ricegaas.dna.affrc.go.jp/) using the PDIL1-1 cDNA sequence as the query. The PDIL1-1 gene, which spans 3,042 bp between the start to stop codons, consists of 10 exons and 9 introns (Fig. 2A). Comparison with the wild type gene sequences showed that each of the three esp2 lines, CM1787, EM44, EM747, contained single nucleotide substitutions. In all lines, the mRNA of the PDIL1-1 gene was not expressed (Takemoto et al. 2002). In CM1787, substitution of an A for a T occurred at nucleotide 194 resulting in the codon change from Lys65 to a termination stop codon in the third exon (Fig. 2B). The reduction of gene expression by nonsense-mediated mRNA decay (NMD) is well documented (Maquat 2004) and is the likely basis for the absence of at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 12 the PDIL1-1 mRNA. Mutations in EM44 and EM747 were the substitution of G to A located at nucleotide positions 2327 and 2435, respectively. These mutations are located at the 3’ end of intron 7 and the 5’ end of intron 8, respectively (Fig. 2B). In both instances, the highly conserved border sequences of the splice sites were disrupted, which would result in incorrect splicing patterns leading to frame shifts or deletions in the mRNA (Brown 1996). These results demonstrate that Esp2 is the structural gene for the PDIL1-1 protein and that its absence is responsible for the abnormal accumulation of proglutelin. PDIL1-1 disrupts the accumulation and packaging of rice seed storage proteins In addition to PDIL1-1 causing changes in proglutelin and mature glutelin subunits, esp2 also affected the levels of the 26 kD α-globulin and prolamines. This effect is readily apparent for the prolamine polypeptide bands at 14 kD and 13 kD which are conspicuously reduced in the nulliplex genotype (Fig. 1A). To obtain more insight on the relationship between storage protein accumulation and the PDIL1-1, the accumulation patterns of glutelin, prolamine and α-globulin were investigated and compared to the temporal accumulation patterns of PDIL1-1 as well as the lumenal chaperone BiP during seed development (Fig. 3). In developing wild type seeds, glutelin acidic and basic subunits are initially detected at 5 days after flowering (DAF) and their levels increase linearly between 10 to 18 DAF (Fig. 3A). Proglutelin and α-globulin levels were low at 5 to 10 DAF but began to increase at 10 DAF. Prolamine polypeptides, at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 13 especially the 14 kD and 16 kD cysteine-rich prolamines, were first detected at 10 DAF and increased significantly after 13 DAF, whereas 13 kD cysteine-poor prolamines started to accumulate somewhat later. By contrast, proglutelin in esp2 developing seeds exhibited a markedly different accumulation pattern. At 5 and 10 DAF, proglutelin levels are very low compared to those of its mature subunits. Hence in young developing seeds, proglutelin is efficiently exported from the ER and transported to PB-II where it is processed into acidic and basic subunits. At 13 DAF and later, however, proglutelin levels increased rapidly and exceeded those seen for individual glutelin acidic and basic subunits. The total amount of proglutelin and its mature subunits were significantly lower in esp2 compared to wild type. The levels of prolamines and α-globulins in esp2 were also significantly reduced, indicating that the deficiency of PDIL1-1 has a general suppressive effect on storage protein expression. Fig. 3C shows the accumulation of PDIL1-1, BiP and the glutelin subunits during the development of wild type seeds. Glutelin acidic and basic subunits are first detected at 6 DAF while PDIL1-1 is detected much earlier at 2 DAF and attains a maximum level at 8 DAF (Fig. 3C). Although BiP was also detected at 2 DAF, its level remained low until 6 DAF where its relative levels increased, attaining a maximum level at 21 DAF. These results readily show that PDIL1-1 is expressed much earlier during seed development than the mature glutelin subunits. at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 14 PDIL1-1 is localized within the cisternal-ER To establish a possible role for PDIL1-1, its intracellular localization in endosperm was investigated using both biochemical and microscopic approaches. Fig. 4 depicts a SDS polyacrylamide gel of fractions obtained from sucrose density gradient centrifugation of protein body-membrane fractions isolated from 15-20 DAF wild type developing seeds. PB-I containing prolamine was enriched in fractions 25 to 29 while PSV containing glutelin was detected in fractions 29 to 33 (Fig. 4A). It was previously shown that proglutelin localized in PB-ER fraction in the esp2 mutant (Takemoto et al. 2002). Immunoblot analysis of the various fractions showed that BiP was distributed throughout the sucrose density gradient and especially prevalent in fractions 1 to 9 and fractions 23 to 29, the latter peak coinciding with prolamine PB-I, which is enriched for this lumenal chaperone (Muench et al. 1997). By contrast, PDIL1-1 was restricted mainly to fractions 1 to 13, which are enriched in light cisternal ER membranes. Very small amounts of PDIL1-1 were detected in PB-I fractions. These results indicate that PDIL1-1 is restricted mainly to the cisternal-ER membranes with very little, if any, associated with prolamine containing PB-I. To verify the location of PDIL1-1 within the cisternal-ER and its exclusion from PB-I, both immunofluorescence and immunoelectron microscopy were performed. As predicted from the sucrose density gradient results, PDIL1-1 label was readily evident over the at China Academ y of Agricultural Sciences on July 13, 2010 http://pcp.oxfordjournals.org D ow nloaded from 15 cisternal ER when viewed by immunofluorescence microscopy (Fig. 5A, B and Supplementary Movie 1). Three-dimensional reconstructions from multiple z sections show that PDIL1-1 label is within the cortical ER network and is almost completely excluded from PB-I (Fig. 5A). Imaging of thin sections from 15 DAF seeds (Fig. 5B) clearly shows the presence of PDIL1-1 in discrete regions of the cisternal-ER immediately adjacent to the prolamine-containing PB-I. Fig. 5C more clearly depicts the relationship between PDIL1-1 and rhodamine signals within the cortical ER, the merged image suggesting that protein bodies are depressed within the surface of the cisternal ER since the central portion of the PB remains magenta in color. Fig. 5D also clearly shows the relationship between PDIL1-1 and BiP, with BiP being localized to the PB-I surface (Muench et al. 1997) whilst PDIL1-1 is present