| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
American Society of Plant Biologists A Novel Membrane Protein That Is Transported to Protein Storage Vacuoles via Precursor-Accumulating Vesicles
a Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 1 To whom correspondence should be addressed. E-mail ihnishi{at}gr.bot.kyoto-u.ac.jp; fax 81-75-753-4141
A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long  -helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum–derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73  ) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.
Protein bodies are widely distributed in the dry seed of higher plants. They are membrane-bound organelles that store various seed proteins. Protein bodies are formed from protein storage vacuoles at the late stage of seed maturation in association with the desiccation of pumpkin seed (Hara-Nishimura et al., 1987  ). Conversely, after seed imbibition, protein bodies swell, start to fuse with one another, and are converted into a large central vacuole (Hara-Nishimura et al., 1991b). The dynamic morphological transformation (i.e., from protein storage vacuoles to protein bodies and then to lytic vacuoles) occurs in the seed cells during seed desiccation and imbibition. Protein bodies can be regarded as a desiccation form of protein storage vacuoles, which are present in dry seed.
The membranes of the protein storage vacuoles and the lytic vacuoles have been isolated from dry seed and seedlings of pumpkin, respectively (Inoue et al., 1995a
A seed-specific water channel,
To elucidate the mechanisms responsible for organellar transformation, it is necessary to clarify what kinds of membrane proteins are specifically expressed and how they are localized on the membranes of protein storage vacuoles during seed maturation. Previously, we found that precursor-accumulating (PAC) vesicles are responsible for the transport of storage proteins directly to protein storage vacuoles (Hara-Nishimura et al., 1998b
At present, it is unclear whether the PAC vesicles are involved in the delivery of proteins other than storage proteins, such as membrane proteins. In this study, we characterized a novel 73-kD membrane protein with a long
Molecular Characterization of a Novel Membrane Protein, MP73, of Protein Storage Vacuoles To characterize membrane components of protein storage vacuoles, protein storage vacuoles were purified from dry pumpkin seed and their membranes were isolated. Five membrane proteins, designated MP23, MP27, MP28, MP32, and MP73, were enriched in the final preparation, although there was some contamination by storage proteins 11S globulin and 7S globulin, as shown in Figure 1 (lanes 1 and 2). Both MP23 and MP28, which were shown previously to be types of TIP water channels (Inoue et al., 1995b), were not extractable from the membranes with an alkaline solution (Figure 1, lane 3). MP27 and MP32 were shown previously to be peripheral membrane proteins (Inoue et al., 1995a). The remaining membrane protein, MP73, is the largest one, and its function is unknown.
To clarify the seed-specific expression of MP73, immunoblotting was performed with antibodies raised against purified MP73 (Figure 1, right). Preimmune serum gave no signal on the blot of 50 µg of total proteins of the isolated membranes of protein storage vacuoles (lane 4). Anti-MP73 antibodies specifically detected MP73 in the membrane fraction (1 µg of total proteins; lane 5) as well as in the isolated membranes of protein storage vacuoles of castor bean (1 µg of total proteins; lane 6). On the immunoblots of various organs (50 µg of total proteins), MP73 was detected in the maturing seed (lane 7) but not in the 10-day-old seedlings (lane 8), the roots (lane 9), the stems (lane 10), or the mature leaves (lane 11). These results suggest that MP73, like TIPs, is a membrane protein that is unique in protein storage vacuoles of various seed. MP73 cDNAs were isolated from the cDNA library of maturing pumpkin cotyledons. The primary structure of the MP73 precursor (685 amino acids) was deduced from the complete nucleotide sequence, as shown in Figure 2A . Figure 2B shows its hydropathy plot. The N-terminal hydrophobic sequence has the features of a putative signal peptide, expected to be cleaved off the C-terminal side of Ala-31 (Figure 2A, open triangle). The sequence of the N-terminal 29 amino acid residues of purified MP73 corresponded to the sequence from Met-245 to Trp-273 (Figure 2A, double underline). Thus, post-translational processing might occur on the C-terminal side of Gly-244 (Figure 2A, closed triangle) that is located in the hydrophilic region of the precursor (Figure 2B). The sequences of 15 peptide fragments obtained from a trypsin digest of purified MP73 were found in the region after Gly-244 (Figure 2A, underlined). These results indicate that the MP73 precursor is composed of a possible 31–amino acid signal peptide for cotranslational insertion into the RER, a 213–amino acid propeptide domain (expected molecular mass, 24 kD [P24]), and a 54-kD domain corresponding to the mature MP73. The calculated molecular mass (54 kD) of the mature molecule is much lower than the 73 kD that was estimated by SDS-PAGE (discussed below).
MP73 has no hydrophobic region sufficient to form a transmembrane domain (Figure 2B), indicating that it is a peripheral membrane protein. This was supported by the result that MP73 was extractable with alkali (Figure 1, lane 3). The C-terminal half of MP73 is a Glu/Arg-rich domain (indicated by the darker shading in Figures 2A and 2B) that is highly hydrophilic and acidic (pI = 4.53). A homology plot shows that 13 repeats are found in the Glu/Arg-rich domain (Figure 2C). A typical repeat sequence is Arg-(Glu)4-Arg-Glu-Arg-(Glu)4-Ala-Arg-Lys (Figure 2D). The Glu/Arg-rich domain is predicted to form a long -helix.
ProMP73 Is Localized on the Membranes of PAC Vesicles, whereas MP73 Is Found on the Membranes of Protein Storage Vacuoles
Four potential N-glycosylation sites were found at positions 296, 334, 366, and 408 of the proMP73 polypeptide (Figure 2A, dots). N-glycosylation is a cotranslational process that occurs in the lumen of the RER. To determine whether proMP73 is glycosylated or not, the purified membranes of the protein storage vacuoles were treated with glycosidases. Treatment of MP73 with either N-glycosidase F or endoglycosidase H reduced its molecular mass as determined by SDS-PAGE, as shown in Figures 4A and 4B . The sensitivity to endoglycosidase H with a substrate specificity toward high mannose oligosaccharides showed that MP73 has high mannose oligosaccharides. An immunoblot with anti–complex glycan antibodies showed no signal for the purified MP73 (Figure 4C, lanes 1 and 2) but a positive signal for the purified PV72 that is known as a glycosylated vacuolar sorting receptor (Figure 4C, lanes 3 and 4). It seems likely that MP73 has no complex glycan. N-glycosylation indicated that MP73 is associated with the lumenal side of the protein storage vacuoles.
PAC Vesicles Mediate the Delivery of Glycosylated ProMP73 to Protein Storage Vacuoles To demonstrate the intracellular transport of proMP73, subcellular fractionation of pulse-chase–labeled maturing seed of castor bean was performed. After the pulse for 30 min, the labeled proMP73 was distributed in the endoplasmic reticulum (ER) and the PAC vesicle fractions (Figure 5A) . The proMP73 disappeared during the 18-hr chase, and the mature MP73 appeared in the supernatant fraction (the S fraction), which contained both the 13% (w/w) sucrose fraction and the interface between 13% (w/w) sucrose and 20% (w/w) sucrose (Figure 5A). Most MP73 protein storage vacuoles were recovered in the S fraction, whereas the crystalloids of protein storage vacuoles were recovered in the pellet fraction (the C fraction). The protein storage vacuoles were ruptured easily by centrifugation because of crystalloids inside the vacuoles (see Figure 8C , top). The membrane fragments from the broken vacuoles might be recovered in the S fraction. A slight amount of MP73 was detected in the C fraction that might contain a few intact vacuoles (Figure 5C, left). These results indicated that proMP73 synthesized on the ER membranes was transported to the PAC vesicle membranes and then to the membranes of protein storage vacuoles, where the precursor might be converted to the mature form.
The effect of tunicamycin on the biosynthesis of MP73 was investigated in the maturing seed. The pulse-labeled seed gave two positive bands corresponding to proMP73 and MP73 (Figure 5B, lane 1). The maturing seed that had been treated with tunicamycin accumulated only the unglycosylated proMP73 (proMP73 ), which has a smaller molecular mass than proMP73 (Figure 5B, lanes 2 and 3). The molecular mass of preproMP73, an in vitro translation product that had a signal peptide but no oligosaccharide, was smaller than that of proMP73 and larger than that of proMP73 (Figure 5B, lanes 3 and 4).
To clarify the effect of tunicamycin on the intracellular transport of proMP73, subcellular fractionation of the pulse-labeled maturing seed that had been incubated in the absence or presence of tunicamycin was performed. After the pulse of tunicamycin-untreated seed, the labeled proMP73 was distributed in the ER and the PAC vesicle fractions, whereas the labeled MP73 was localized in the S fraction and slightly in the C fraction (Figure 5C, left). On the other hand, tunicamycin treatment of the maturing seed resulted in the efficient deposition of proMP73
A Soluble Proteinase Is Involved in the Processing of ProMP73 to Form a 100-kD Complex in Protein Storage Vacuoles
To clarify the higher structure of MP73, the isolated membranes of the protein storage vacuoles were incubated with a cross-linking reagent, dithiobissuccinimidylpropionate (DSP). As shown in Figure 7 , the level of a 100-kD complex was increased in parallel with the increased concentration of DSP on the immunoblot with anti-MP73 antibodies (lanes 1 to 3). When the cross-linkage of the complex was broken with a reducing agent, only MP73 was detected on the blot (lanes 4 to 6). This result indicates that MP73 binds to an unknown molecule to form a 100-kD complex on the membranes of protein storage vacuoles. Because the molecular mass of the complex was similar to that of proMP73, it is possible that the 24-kD domain (P24) remains associated with MP73 after post-translational processing.
MP73 on the Membranes of Protein Storage Vacuoles Appeared during Seed Maturation and Disappeared after Seed Germination, in Association with the Morphological Changes of Protein Storage Vacuoles Figure 8A shows the developmental change in the levels of the storage proteins (7S and 11S globulin) during seed maturation followed by seed germination. The levels of storage proteins increased from 18 to 38 days after anthesis and reached a maximum in the dry seed. A drastic degradation of the storage proteins occurred between 2 and 4 days after germination. Figure 8B shows the change in the level of MP73. The results show that MP73 is transiently accumulated in protein storage vacuoles specifically during seed desiccation and is degraded after seed germination. Protein storage vacuoles accumulating storage proteins are converted into the desiccation form (protein bodies) after seed desiccation (Hara-Nishimura et al., 1987 ), as shown in Figure 8C. The protein bodies are fused to each other and give rise to lytic vacuoles (Hara-Nishimura et al., 1991b). The increase in the abundance and decline of MP73 on the membranes is associated with the morphological change of the organelles, as shown in Figure 8C.
PAC Vesicles Are Responsible for the Transport of a Membrane Protein Together with Storage Proteins to Protein Storage Vacuoles Previously, we demonstrated that various seed storage proteins form an aggregate within the lumen of the ER and that the insoluble aggregate is transported directly to protein storage vacuoles via PAC vesicles (Hara-Nishimura et al., 1998b ). Our results indicate that the PAC vesicles are involved in the delivery of not only storage proteins but also a membrane protein (Figures 3 and 5). When the seed were treated with tunicamycin, an inhibitor of N-glycosylation on ER membranes, we found efficient deposition of the unglycosylated proMP73 (proMP73) in the PAC vesicles (Figure 5C). In contrast, we found no accumulation of MP73 in the vacuoles (Figure 5C). The failure of the accumulation of the mature MP73 suggests two possibilities. One is that proMP73 is not delivered from the PAC vesicles to the vacuoles. Another is that proMP73 is unstable enough to be degraded in the vacuoles.
Two other peripheral membrane proteins, MP27 and MP32, share a similar sequence with MP73 (discussed below). It is possible that they are transported to the membranes of protein storage vacuoles in a PAC-dependent pathway, as is MP73. Jiang and Rogers (1998)
Processing and Oligomerization of MP73 on the Vacuolar Membranes In contrast to MP27 and MP32, proMP73 was post-translationally processed at the C-terminal side of Gly-244 (Figure 2A), which is not consistent with the substrate specificity of VPEs. We found that there was processing activity to convert proMP73 to MP73 in a vacuolar subcellular fraction (Figure 6). Thus, another proteinase in the vacuoles is responsible for the maturation of MP73 on the membranes. It is possible that after processing at Gly-244, the N-terminal P24 domain is still attached to the MP73 domain to form a 100-kD complex and that the P24 domain exerts some functions on the membranes of protein storage vacuoles after being cleaved off from the precursor protein. MP27 and MP32 are produced similarly from a single precursor, and they form a complex on vacuolar membranes (K. Inoue, M. Nishimura, and I. Hara-Nishimura, unpublished data).
Molecular Characterization of the Glu/Arg-Rich Domain of MP73
The molecular mass of the MP73 polypeptide was deduced to be 53,911 D. The molecular mass of the fully glycosylated MP73 could be
MP73 Functions on the Membranes of Protein Storage Vacuoles of Seed during Desiccation and Imbibition
Late embryogenesis–abundant proteins have been implicated in the acquisition of desiccation tolerance of maturing seed (Ingram and Bartels, 1996 MP73 is a novel membrane protein, but a limited region of 59 amino acids from Asn-259 to Val-317 shows high homology with a part of pumpkin MP32 (49%) and proteins with unknown function of carrot (GenBank accession number U47078; 53%) and Arabidopsis (GenBank accession number Z48554; 53%). In contrast, P24 exhibits very low homology with the corresponding N-terminal domains of MP27 (23%), the carrot protein (23%), and the Arabidopsis protein (30%). Further analysis with a knockout mutant of Arabidopsis will help to clarify the function of such peripheral membrane proteins on the membranes.
Plant Materials Seed of pumpkin (Cucurbita maxima cv Kurokawa Amakuri Nankin) were purchased from Aisan Shubyo Seed Co. (Nagoya, Japan), and seed of castor bean (Ricinus communis) were a gift from Ito Oil Co. (Yokkaichi, Japan). The seed were used directly for isolation of protein storage vacuoles. To obtain germinating seed and growing seedlings, the seed were soaked in water for 16 hr and then grown in the dark at 25°C for up to 6 days for pumpkin. To obtain immature seed, pumpkin seed were grown at the experimental farm of the National Institute for Basic Biology during the summer season. Cotyledons from maturing seed were freshly harvested at 18 to 38 days after anthesis to use for experimentation. Castor bean seed also were grown in a greenhouse, and endosperm from maturing seed were freshly harvested at 20 days after anthesis. Whole homogenates of pumpkin seed at various stages of seed development and germination were subjected to SDS-PAGE and subsequent immunoblot analysis, as were the homogenates of roots, stems, and mature leaves, as described previously (Hatano et al., 1997 ).
Isolation of Protein Storage Vacuoles and Preparation of Protein Storage Vacuole Membranes
The vacuolar membranes were prepared from the isolated protein storage vacuoles of pumpkin and castor bean, as described previously (Mettler and Beevers, 1979
To detect integral membrane proteins, the membranes of the isolated protein storage vacuoles were extracted with alkali as described previously (Mäder and Chrispeels, 1984
Isolation of Precursor-Accumulating Vesicles
Determination of Both N-Terminal and Internal Amino Acid Sequences
To determine the internal sequences, the band corresponding to MP73 on an SDS gel with Coomassie Brilliant Blue R 250 was subjected to in-gel digestion as described by Hellman et al. (1995)
Isolation of MP73 cDNAs and Determination of Nucleotide Sequences The two isolated cDNAs of 1061 and 1012 bp did not cover the entire sequence of MP73. Subsequently, we amplified DNAs covering the 5' and 3' regions of MP73 cDNA using a 5'-Full RACE (rapid amplification of cDNA ends) Core Set and a 3'-Full RACE Core Set (Takara, Tokyo, Japan), respectively. The 5'-RACE gave a 202-bp DNA that contained an initiation codon. The 3'-RACE gave a 978-bp DNA that contained a poly(A) tail.
DNA sequencing was performed with a DNA sequencer (model 377; Applied Biosystems) and 21M13 forward and M13 reverse fluorescent primers according to the manufacturer's directions. The nucleotide and deduced amino acid sequences were analyzed with DNA analytical software (GeneWorks; IntelliGenetics, Mountain View, CA). The hydropathy profile of the amino acid sequence was computed by application of the algorithms of Kyte and Doolittle (1982)
Specific Antibodies
Immunoblot Analysis
Electron Microscopic Analysis by High Pressure Freezing, Freeze Substitution, and Immunogold Labeling
Immunogold labeling procedures were essentially the same as those described previously (Nishimura et al., 1993
Procedures for ultrastructural studies of chemically fixed samples, the maturing seed, dry seed, and 4-day-old seed of pumpkin were essentially the same as those described previously (Hara-Nishimura et al., 1993a
In Vivo Pulse-Chase Labeling To examine the effect of tunicamycin on the glycosylation of proMP73, maturing castor bean seed were incubated in the absence or presence of 3 or 30 µg/mL tunicamycin for 3 hr before a 4-hr pulse labeling, as described above. The labeled proteins were immunoprecipitated with anti–castor bean MP73 homolog antibodies and subjected to SDS-PAGE and fluorography. For subcellular fractionation, maturing castor bean seed were incubated in the absence or presence of 30 µg/mL tunicamycin for 3 hr before a 4-hr pulse labeling. The tissues were subjected to subcellular fractionation as described above. Each fraction was immunoprecipitated with anti–castor bean MP73 homolog antibodies and protein A–Sepharose. The precipitate was subjected to SDS-PAGE and fluorography.
In Vitro Translation
In Vitro Conversion of ProMP73 to MP73
Treatment with N-Glycosidase F and Endoglycosidase H
Cross-Linking with Dithiobissuccinimidylpropionate
We thank Chiyeko Namba for growing castor bean and pumpkin plants. We are grateful to Yumiko Makino for helpful support with peptide sequencing. We also thank Yuka Takeuchi and Maki Kondo for helpful support with electron microscopy. This work was supported by Grants-in-Aid for the Human Frontier Science Program (RG0018/2000-M 103) and the Research for the Future Program (JSPS-RFTF96L60407) and by the Japan Society for the Promotion of Science and for Scientific Research (Grant No. 12304049) from the Ministry of Education, Science, Sports, and Culture of Japan. Received May 2, 2001; accepted July 11, 2001.
Chou, P.Y., and Fasman, G.D. (1974). Prediction of protein conformation. Biochemistry 13, 222–245.[CrossRef][Medline] Craig, S., and Staehelin, L.A. (1998). High pressure freezing of intact plant tissues: Evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems. Eur. J. Cell Biol. 46, 80–93.
Hara-Nishimura, I., and Nishimura, M. (1987). Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons. Plant Physiol. 85, 440–445.
Hara-Nishimura, I., Nishimura, M., Matsubara, H., and Akazawa, T. (1982). Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin. Plant Physiol. 70, 699–703.
Hara-Nishimura, I., Nishimura, M., and Akazawa, T. (1985). Biosynthesis and intracellular transport of 11S globulin in developing pumpkin cotyledons. Plant Physiol. 77, 747–752. Hara-Nishimura, I., Hayashi, M., Nishimura, M., and Akazawa, T. (1987). Biogenesis of protein bodies by budding from vacuoles in developing pumpkin cotyledons. Protoplasma 136, 49–55.[CrossRef] Hara-Nishimura, I., Inoue, K., and Nishimura, M. (1991a). A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett. 294, 89–93.[CrossRef][ISI][Medline] Hara-Nishimura, I., Inoue, K., and Nishimura, M. (1991b). Reversible transformation between vacuoles and protein bodies in pumpkin cotyledons. In Molecular Approaches to Compartmentation and Metabolic Regulation, A. Huang and L. Taiz, eds (American Society of Plant Physiologists, Baltimore, MD), pp. 245–246. Hara-Nishimura, I., Takeuchi, Y., Inoue, K., and Nishimura, M. (1993a). Vesicle transport and processing of the precursor to 2S albumin in pumpkin. Plant J. 4, 793–800.[CrossRef][ISI][Medline] Hara-Nishimura, I., Takeuchi, Y., and Nishimura, M. (1993b). Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni. Plant Cell 5, 1651–1659.[Abstract] Hara-Nishimura, I., Shimada, T., Hiraiwa, N., and Nishimura, M. (1995). Vacuolar processing enzyme responsible for maturation of seed proteins. J. Plant Physiol. 145, 632–640. Hara-Nishimura, I., Kinoshita, T., Hiraiwa, N., and Nishimura, M. (1998a). Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles. J. Plant Physiol. 152, 668–674.[ISI]
Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y., and Nishimura, M. (1998b). Transport of storage proteins to protein-storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell 10, 825–836.
Hatano, K., Shimada, T., Hiraiwa, N., Nishimura, M., and Hara-Nishimura, I. (1997). A rapid increase in the level of binding protein (BiP) is accompanied by synthesis and degradation of storage proteins in pumpkin cotyledons. Plant Cell Physiol. 38, 344–351.
Hayashi, Y., and Ueda, K. (1987). Localization of mannose, N-acetylglucosamine and galactose in the Golgi apparatus, plasma membranes and cell walls of Scenedesmus acuminatus. Plant Cell Physiol. 28, 1357–1362. Hellman, U., Wernstedt, C., Góñez, J., and Heldin, C.-H. (1995). Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451–455.[CrossRef][ISI][Medline]
Hinz, G., Hilmer, S., Baumer, M., and Hohl, I. (1999). Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the Golgi apparatus of developing pea cotyledons in different transport vesicles. Plant Cell 11, 1509–1524.
Hiraiwa, N., Takeuchi, Y., Nishimura, M., and Hara-Nishimura, I. (1993). A vacuolar processing enzyme in maturing and germinating seeds: Its distribution and associated changes during development. Plant Cell Physiol. 34, 1197–1204. Hiraiwa, N., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (1997a). An aspartic proteinase is involved in the maturation of storage proteins in concert with the vacuolar processing enzyme. Eur. J. Biochem. 246, 133–141.[ISI][Medline] Hiraiwa, N., Nishimura, M., and Hara-Nishimura, I. (1997b). Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae. Plant J. 12, 819–829.[CrossRef][ISI][Medline] Improta-Brears, T., Ghosh, S., and Bell, R.M. (1999). Mutational analysis of Raf-1 cysteine rich domain: Requirement for a cluster of basic amino acids for interaction with phosphatidylserine. Mol. Cell. Biochem. 198, 171–178.[CrossRef][Medline] Ingram, J., and Bartels, D. (1996). The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377–403.[CrossRef][ISI][Medline] Inoue, K., Motozaki, A., Takeuchi, Y., Nishimura, M., and Hara-Nishimura, I. (1995a). Molecular characterization of proteins in protein-body membranes that disappear most rapidly during transformation of protein bodies into vacuoles. Plant J. 7, 235–243.[CrossRef][ISI][Medline] Inoue, K., Takeuchi, Y., Nishimura, M., and Hara-Nishimura, I. (1995b). Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds. Plant Mol. Biol. 28, 1089–1101.[CrossRef][ISI][Medline]
Jiang, L., and Rogers, J.C. (1998). Integral membrane protein sorting to vacuoles in plant cells: Evidence for two pathways. J. Cell Biol. 143, 1183–1199.
Johnson, K.D., Herman, E.M., and Chrispeels, M.J. (1989). An abundant, highly conserved tonoplast protein in seeds. Plant Physiol. 91, 1006–1013. Koumoto, Y., Shimada, T., Kondo, M., Hara-Nishimura, I., and Nishimura, M. (1999). Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana. Plant J. 17, 467–477.[CrossRef][ISI][Medline] Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.[CrossRef][ISI][Medline] Mäder, M., and Chrispeels, M.J. (1984). Synthesis of an integral membrane protein of the protein-body membrane in Phaseolus vulgaris cotyledons. Planta 160, 330–340.[CrossRef] Maeshima, M., Hara-Nishimura, I., Takeuchi, Y., and Nishimura, M. (1994). Accumulation of vacuolar H+-pyrophosphatase and H+-ATPase during reformation of the central vacuole in germinating pumpkin seeds. Plant Physiol. 106, 61–69.[Abstract] Melroy, D.L., and Herman, E.M. (1991). TIP, an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon, undergoes developmentally regulated membrane accumulation and removal. Planta 184, 113–122.
Mettler, I.J., and Beevers, H. (1979). Isolation and characterization of the protein body membrane of castor beans. Plant Physiol. 64, 506–511. Nishimura, M., Takeuchi, Y., De Bellis, L., and Hara-Nishimura, I. (1993). Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons. Protoplasma 175, 131–137.[CrossRef]
Pearson, W.R., and Lipman, D.J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444–2448.
Shimada, T., Hiraiwa, N., Nishimura, M., and Hara-Nishimura, I. (1994). Vacuolar processing enzyme of soybean that converts proprotein to the corresponding mature forms. Plant Cell Physiol. 35, 713–718. Strzalka, K., Hara-Nishimura, I., and Nishimura, M. (1995). Changes in physical properties of vacuolar membrane during transformation of protein bodies into vacuoles in germinating pumpkin seeds. Biochim. Biophys. Acta 1239, 103–110.[Medline]
Wada, I., Rindress, D., Cameron, P.H., Ou, W.-J., Doherty, J.J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D.Y. and Bergeron, J.J.M. (1991). SSRa and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem. 266, 19599–19610.
Yamada, K., Shimada, T., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (1999). Multiple functional proteins are produced by cleaving Asn-Gln bonds of a single precursor by vacuolar processing enzyme. J. Biol. Chem. 274, 2563–2570.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
Abstract
INTRODUCTION
TIP is newly synthesized and incorporated into the vacuolar membranes after seed germination (Maeshima et al., 1994
64 kD, which is much lower than the value of 73 kD estimated from SDS-PAGE (