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Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

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Abstract

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope “himet” (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.

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Abbreviations

ER:

endoplasmic reticulum

BiP:

binding protein

IgG:

immunoglobulin G

Mr :

relative molecular mass

References

  • Akasofu, H., Yamauchi D., Mitsuhashi, W., Minamikawa, T. (1989) Nucleic acid sequence of cDNA sulfhydryl-endopeptidase (SHEP) from cotyledons of germinating Vigna mungo seeds. Nucleic Acids Res. 17, 6733

    Google Scholar 

  • Baumgartner, B., Chrispeels, M.J. (1977) Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung bean seedlings. Eur. J. Biochem. 77, 223–233

    Google Scholar 

  • Baumgartner, B., Tokuyasu, K.T., Chrispeels, M.J. (1978) Localization of vicilin peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescence microscopy. J. Cell Biol. 79, 10–19

    Google Scholar 

  • Blagrove, R.J., Lilley, G.G., Van Donkelaar, A., Sun, S.M. Hall, T.C. (1984) Structural studies of a french bean storage protein: phaseolin. Int. J. Biol. Macromol. 6, 137–141

    Google Scholar 

  • Bollini, R., Vitale, A., Chrispeels, M.J. (1983) In vitro and in vivo processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps. J. Cell Biol. 96, 999–1007

    Google Scholar 

  • Bonifacino, J.S., Lippincott-Schwartz, J. (1991) Degradation of proteins within the endoplasmic reticulum. Curr. Opin. Cell Biol 3, 592–600

    Google Scholar 

  • Chrispeels, M.J. (1983) The Golgi apparatus mediates the transport of phytohemagglutinin to the protein bodies in bean cotyledons. Planta 158, 140–151

    Google Scholar 

  • Chrispeels, M.J. (1991) Sorting of proteins in the secretory system. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 1–24

    Google Scholar 

  • D'Amico, L., Valsasina, B., Daminati, M.G., Fabbrini, M.S., Nitti, G., Bollini, R., Ceriotti, A., Vitale, A. (1992) Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum. Plant J. 2, 443–455

    Google Scholar 

  • Denecke, J., De Rycke, R., Botterman, J. (1992) Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. EMBO J. 11, 2345–2355

    Google Scholar 

  • Denecke, J., Goldman, M.H.S., Demolder, J., Seurinck, J., Botterman, J. (1991) The tobacco luminal binding protein is encoded by a multigene family. Plant Cell 3, 1025–1035

    Google Scholar 

  • Greenwood, J.S., Chrispeels, M.J. (1985) Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco. Plant Physiol. 79, 65–71

    Google Scholar 

  • Hara-Nishimura, I., Takeuchi, Y., Nishimura, M. (1993) Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma manoni. Plant Cell 5, 1651–1659

    Google Scholar 

  • Herman, E.M. (1994) Multiple origins of intravacuolar protein accumulation of plant cells. Adv. Struct. Biol. 3, 243–283

    Google Scholar 

  • Herman, E.M., Tague, B.W., Hoffman, L.M., Kjemtrup, S.E., Chrispeels, M.J. (1990) Retention of phytohemagglutinin with carboxyterminal KDEL in the nuclear envelope and the endoplasmic reticulum. Planta 182, 305–312

    Google Scholar 

  • Hiraiwa, N., Takeuchi, Y., Nishimura, M., Hara-Nishimura, I. (1993) A vacuolar processing enzyme in maturing and germinating seed: its distribution and associated changes during development. Plant Cell Physiol. 34, 1197–1204

    Google Scholar 

  • Hoffman L.M., Donaldson, D.D., Herman, E.M. (1988) A modified storage protein is synthesized, processed and degraded in the seeds of transgenic plants. Plant Mol. Biol. 11, 717–729

    Google Scholar 

  • Höfgen, R., Willmitzer, L. (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16, 9877

    Google Scholar 

  • Höfte, H., Chrispeels, M.J. (1992) Protein sorting to the vacuolar membrane. Plant Cell 4, 995–1004

    Google Scholar 

  • Hurtley, S.M., Bole, D.G., Hoover-Litty, H., Helenius, A., Copeland, C.S. (1989) Interactions of misfolded influenza virus hemaglutinin with binding protein (BiP). J. Cell Biol. 108, 2117–2126

    Google Scholar 

  • Johnson, E.d., Knight, J., Gayler, K.R. (1985) Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin). Biochem. J. 232, 673–679

    Google Scholar 

  • Jones, A.M., Herman, E.M. (1993) A KDEL-containing auxin-binding protein is secreted to the plasma membrane and cell wall. Plant Physiol. 101, 595–606

    Google Scholar 

  • Kalinski, A., Melroy, D.L., Dwivedi, R.S., Herman, E.M. (1992) A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation. J. Biol. Chem. 267, 12068–12076

    Google Scholar 

  • Kassenbrock, C.K., Garcia, P.D., Walter, P., Kelly, R.B. (1988) Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333, 90–93

    Google Scholar 

  • Klausner, R.D., Sitia, R. (1991) Protein degradation in the endoplasmic reticulum. Cell 62, 611–614

    Google Scholar 

  • Kozutsumi, Y., Segal, M., Normington, K., Slaughter, C., Gething, M., Sambrook, J. (1988) The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-reguated proteins. Nature 332, 461–464

    Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

    PubMed  Google Scholar 

  • Lawrence, M.C., Suzuki, E., Varghese, J.N., Davis, P.C., Van Donkelaar, A., Tulloch, P.A., Colman, P.M. (1990) The threedimensional structure of the seed storage protein phaseolin at 3 Å resolution. EMBO J. 9, 9–15

    Google Scholar 

  • Lee, H.I., Gal, S., Newman, T.C, Raikhel, N.V. (1993) The Arabidopsis endoplasmic reticulum retention receptor functions in yeast. Proc. Natl. Acad. Sci. USA 90, 11433–11437

    Google Scholar 

  • Lippincott-Schwartz, J., Bonifacino, J.S., Yuan, L.C., Klausner, R.D. (1988) Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell 54, 209–220

    Google Scholar 

  • Lodish, H.F. (1988) Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A ratelimiting step in protein maturation and secretion. J. Biol. Chem. 263, 2107–2110

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275

    CAS  PubMed  Google Scholar 

  • Machamer, C.E., Doms, R.W., Bole, D.G., Helenius, A., Rose, J.K. (1990) Heavy chain binding protein recognizes incompletely dislfide-bonded forms of vesicular stomatitis virus G Protein. J. Biol. Chem. 265, 6879–6883

    Google Scholar 

  • Machamer, C.E., Rose, J.K. (1988) Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. J. Biol Chem. 263, 5948–5954

    Google Scholar 

  • Marquardt, T., Helenius, A. (1992) Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum. J. Cell Biol. 117, 505–513

    Google Scholar 

  • Mitsuhashi, W., Koshiba, T., Minamikawa, T. (1986) Separation and characterization of two endopeptidases from cotyledons of germinating Vigna mungo seeds. Plant Physiol. 80, 628–634

    Google Scholar 

  • Mitsuhashi, W., Minamikawa, T. (1989) Synthesis and posttranslational activation of sulfhydryl-endopeptidase in cotyledons of germinating Vigna mungo seeds. Plant Physiol. 82, 274–279

    Google Scholar 

  • Munro, S., Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907

    Google Scholar 

  • Napier, R.M., Fowke, L.C., Hawes, C., Lewis, M., Pelham, H.R.B. (1992) Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J. Cell Sci. 102, 261–271

    Google Scholar 

  • Nielsen, N.C., Jung, R., Nam, Y.W., Beaman, T.W., Olivera, L.O. (1994) Synthesis and assembly of 11S globulins. In: Proc. Intl. Workshop on Life Science in Production and Food Consumption of Agricultural Products, Cherry, J.P., Kobuyshia, eds. Tsukaba, Japan, in press

  • Okamoto, T., Nakayama, H., Seta, K., Isobe, T., Minamikawa, T. (1994) Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cyseine endopeptidase (SH-EP). FEBS Lett., in press

  • Pedrazzini, E., Giovinazzo, G., Bollini, R., Ceriotti, A., Vitale, A. (1994) Binding of BiP to an assembly-defective protein in plant cells. Plant J. 5, 103–110

    Google Scholar 

  • Scott, M.P., Jung, R., Muntz, K., Nielsen, N.C. (1992) A protease responsible for post-translational cleavage of a conserved AsnGly linkage in glycinin, the major seed storage protein of soybean. Proc. Natl. Acad. Sci. USA 89, 658–662

    Google Scholar 

  • Stafford, F.J., Bonifacino, J.S. (1991) A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation. J. Cell Biol. 115, 1225–1236

    Google Scholar 

  • Stanssens, P., Opsomer, C., McKeown, Y.M., Kramer, W., Zabeau, M., Fritz, H.-J, (1989) Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 17, 4441–4454

    Google Scholar 

  • Sturm, A., van Kuik, J.A., Vliegenthart, J.F.G., Chrispeels, M.J. (1987) Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin. J. Biol. Chem. 262, 13392–13403

    Google Scholar 

  • Sturm, A., Voelker, T.A., Herman, E.M., Chrispeels, M.J. (1988) Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco. Planta 175, 170–183

    Google Scholar 

  • Tanaka, T., Minamikawa, T., Yamauchi, D., Ogushi, Y. (1993) Expression of an endopeptidase (EP-C1) in Phaseolus vulgaris plants. Plant Physiol. 101, 421–428

    Google Scholar 

  • Tanaka, T., Yamauchi, D., Minamikawa, T. (1991) Nucleotide sequence of cDNA for an endopeptidase (EP-C1) from pods of maturing Phaseolus vulgaris fruits. Plant Mol. Biol. 16, 1083–1084

    Google Scholar 

  • Van der Wilden, W., Gilkes, N., Chrispeels, M.J. (1980a) The endoplasmic reticulum of mungbean cotyledons: role in the accumulation of hydrolases in protein bodies during seedling growth. Plant Physiol. 66, 390–394

    Google Scholar 

  • Van der Wilden, W., Herman, E.M., Chrispeels, M.J. (1980b) Protein bodies of mung bean cotyledons as autophagic organelles. Proc. Natl. Acad. Sci. USA 77, 428–432

    Google Scholar 

  • Voelker, T., Sturm, A., Chrispeels, M.J. (1987) Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco. EMBO J. 6, 3571–3577

    Google Scholar 

  • Voelker, T.A., Herman, E.M., Chrispeels, M.J. (1989) In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability. Plant Cell 1, 95–104

    Google Scholar 

  • Wandelt, C.I., Khan, M.R.I., Craig, S., Schroeder, H.E., Spencer, D.; Higgins, T.J.V. (1992) Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J. 2, 181–192

    Google Scholar 

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Author notes

  1. José J. Pueyo

    Present address: CSIC, Apartado 202, 50080, Zaragoza, Spain

Authors and Affiliations

  1. Department of Biology, University of California, San Diego, 9500 Gilman Drive, 92093-0116, La Jolla, CA, USA

    José J. Pueyo & Maarten J. Chrispeels

  2. Plant Molecular Biology Laboratory, USDA/ARS, 20705, Beltsville, MD, USA

    Eliot M. Herman

Authors
  1. José J. Pueyo
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  2. Maarten J. Chrispeels
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  3. Eliot M. Herman
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The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentioned

This work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research.

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Pueyo, J.J., Chrispeels, M.J. & Herman, E.M. Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole. Planta 196, 586–596 (1995). https://doi.org/10.1007/BF00203660

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