Identification of a Membrane-associated Cysteine Protease with Possible Dual Roles in the Endoplasmic Reticulum and Protein Storage Vacuole
2001; Elsevier BV; Volume: 276; Issue: 1 Linguagem: Inglês
10.1074/jbc.m003078200
ISSN1083-351X
AutoresTakashi Okamoto, Kiminori Toyooka, Takao Minamikawa,
Tópico(s)Pineapple and bromelain studies
ResumoSH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associatedcysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed. SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associatedcysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed. endoplasmic reticulum protein storage vacuoles polyacrylamide gel electrophoresis membrane-associated cysteine protease The endoplasmic reticulum (ER)1 is the port of entry of proteins into the endomembrane system. In this organelle, there are a number of soluble proteins, membrane proteins, and molecular chaperones which are involved in folding, glycosylation, assembly, and maturation of nascent proteins (1Galili G. Sengupta-Gopalan C. Ceriotti A Plant Mol. Biol. 1998; 38: 1-29Crossref PubMed Google Scholar, 2Okita T.W. Rogers J.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 327-350Crossref PubMed Scopus (141) Google Scholar, 3Vital A. Denecke J. Plant Cell. 1999; 11: 615-628PubMed Google Scholar). The soluble proteins localized in the lumen of the ER have a retention signal, KDEL or HDEL, at the C terminus (4Munro S. Pelham H.B.R. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1562) Google Scholar, 5Pelham H.B.R. Annu. Rev. Cell Biol. 1989; 5: 1-23Crossref PubMed Scopus (539) Google Scholar, 6Napier R.M. Fowke L.C. Hawes C.R. Lewis M. Pelham H.B.R. J. Cell Sci. 1992; 102: 261-271Crossref PubMed Google Scholar), and this signal is known to be recognized by the K(H)DEL receptor on the Golgi complex, which mediates retrieving K(H)DEL-tailed proteins to the ER. The molecular mechanisms of the ER retention of soluble proteins are conserved through animal, plant, and yeast cells (7Lee H.I. Gal S. Newman T.C. Raikhel N.V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11433-11437Crossref PubMed Scopus (91) Google Scholar, 8Denecke J. De Rycke R. Botterman J. EMBO J. 1992; 11: 2345-2355Crossref PubMed Scopus (231) Google Scholar, 9Herman E.M. Tague B.W. Hoffman L.M. Kjemtrup S.E. Chrispeels M.J. Planta. 1990; 182: 305-312Crossref PubMed Scopus (69) Google Scholar).In several kinds of plants, unique papain-type proteinases possessing a C-terminal KDEL sequence have been identified (10Akasofu H. Yamauchi D. Mitsuhashi W. Minamikawa T. Nucleic Acids Res. 1989; 17: 6733Crossref PubMed Scopus (59) Google Scholar, 11Becker C. Shutov A.D. Nong V.H. Senyuk V.I. Jung R. Horstmann C. Fischer J. Nielsen N.C. Müntz K. Eur. J. Biochem. 1995; 228: 456-462Crossref PubMed Scopus (82) Google Scholar, 12Guerrero C. de la Calle M. Reid M.S. Valpuesta V. Plant Mol. Biol. 1998; 36: 565-571Crossref PubMed Scopus (102) Google Scholar, 13Tanaka T. Yamauchi D. Minamikawa T. Plant Mol. Biol. 1991; 16: 1083-1084Crossref PubMed Scopus (47) Google Scholar, 14Valpuesta V. Lange N.E. Guerrero C. Reid M.S. Plant Mol. Biol. 1995; 28: 575-582Crossref PubMed Scopus (109) Google Scholar, 15Schmid M. Simpson D. Kalousek F. Gietl C. Planta. 1998; 206: 466-475Crossref PubMed Scopus (77) Google Scholar, 16Cercos M. Santamaria S. Carbonell J. Plant Physiol. 1999; 119: 1341-1348Crossref PubMed Scopus (53) Google Scholar). One such KDEL-tailed cysteine protease, designated SH-EP, was first isolated from cotyledons of germinated Vigna mungo seeds as the enzyme responsible for degradation of storage proteins accumulated in protein storage vacuoles (PSV) of cotyledon cells (10Akasofu H. Yamauchi D. Mitsuhashi W. Minamikawa T. Nucleic Acids Res. 1989; 17: 6733Crossref PubMed Scopus (59) Google Scholar, 17Mitsuhashi W. Koshiba T. Minamikawa T. Plant Physiol. 1986; 80: 628-634Crossref PubMed Google Scholar). SH-EP is synthesized on membrane-bound ribosomes as a 43-kDa precursor through co-translational cleavage of the signal peptide, and the precursor is processed to the 33-kDa mature enzyme via 39- and 36-kDa intermediates during or after transport to the vacuoles (18Mitsuhashi W. Minamikawa T. Plant Physiol. 1989; 89: 274-279Crossref PubMed Scopus (65) Google Scholar, 19Okamoto T. Nakayama H. Seta K. Isobe T. Minamikawa T. FEBS Lett. 1994; 351: 31-34Crossref PubMed Scopus (33) Google Scholar).The function of the KDEL-tail on a cysteine protease whose final destination is the PSV has been an interesting question. Based on analysis of the heterologous expression of SH-EP and a KDEL deletion mutant of SH-EP in insect Sf9 cells and subcellular fractionation of cotyledon cells, it was proposed that the KDEL-tail of SH-EP functions to store the enzyme as a transient zymogen in the ER, and that the conversion of the 43-kDa SH-EP into the 42-kDa form in/at the ER is accompanied by the removal of the C-terminal propeptide containing the KDEL-tail (20Okamoto T. Minamikawa T. Edward C. Vakharia V. Herman E. J. Biol. Chem. 1999; 274: 11390-11398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). From these observations, the protease responsible for removal of the C-terminal propeptide of SH-EP has been supposed to exist in the lumen of the ER (20Okamoto T. Minamikawa T. Edward C. Vakharia V. Herman E. J. Biol. Chem. 1999; 274: 11390-11398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Recently, Toyooka et al. (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar) showed that in cotyledon cells of V. mungo seedlings, a proform of SH-EP synthesized in the ER accumulated at the edge or middle regions of the ER where the transport vesicle was formed. The vesicle, containing a large amount of pro-SH-EP, termed KV, budded off from the ER, bypassed the Golgi complex, and fused to PSV (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar). It was proposed that the KDEL-tail of SH-EP functions as the signal for accumulation of pro-SH-EP at the edge or middle regions of the ER where formation of KV proceeds (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar).In this study, the protease involved in the processing of 43-kDa SH-EP (KDEL-attached form) into 42-kDa SH-EP (KDEL-removed form) was purified to homogeneity from microsomes of cotyledons of V. mungoseedlings, and a cDNA clone for the enzyme was isolated. Immunocytochemical and sucrose gradient analyses of cotyledon cells were carried out to determine the intracellular localization of the protease. The protease was also expressed in insect Sf9 cells in order to observe the characteristics of the membrane association of the enzyme. The possible dual functions of this enzyme in cells will be discussed.DISCUSSIONAlthough MCP was initially isolated as a protease which is involved in post-translational cleavage of the C-terminal propeptide of SH-EP containing the KDEL-tail within the ER, immunocytochemical analysis of cotyledon cells revealed that MCP is localized in PSV as well as the ER, and the amino acid sequence of MCP deduced from its cDNA indicated that the enzyme is a member of papain family which are potentially degradative enzymes. The involvement of MCP in degradation of storage proteins is consistent with the report that an MCP homologue from Vicia faba was isolated as an enzyme responsible for globulin hydrolysis in cotyledons of germinated V. faba seeds (30Yu W.J. Greenwood J.S. J. Exp. Bot. 1994; 271: 261-268Crossref Scopus (25) Google Scholar, 31Yu W.J. Greenwood J.S. Plant Physiol. PGR. 1996; 112: 862Google Scholar). It has been shown that there are some kinds of proteinases in the cotyledons of germinated V. mungo seeds (17Mitsuhashi W. Koshiba T. Minamikawa T. Plant Physiol. 1986; 80: 628-634Crossref PubMed Google Scholar, 28Okamoto T. Minamikawa T. Plant Mol. Biol. 1999; 39: 63-73Crossref PubMed Scopus (51) Google Scholar) and other papain-type proteinases are thought to exist in the cotyledons, however, only MCP showed the proteolytic activity that converts the recombinant 43-kDa SH-EP (C152G) to the 42-kDa form. In addition, general cysteine proteinase, papain, cleaved the recombinant 43-kDa SH-EP (C152G) to the 33-kDa form through removal of the N-terminal prosequence of the proenzyme. This processing profile is apparently different from that of the recombinant 43-kDa SH-EP (C152G) by MCP. This suggest the possibility that the processing activity for the conversion of the recombinant 43-kDa SH-EP (C152G) to the 42-kDa form is unique to MCP.The present results of sucrose gradient centrifugation and immunocytochemistry of cotyledon cells suggest the possibility that MCP exists as an active mature enzyme in the ER in association with the membrane, although papain-type proteases have not been expected to be localized in the ER lumen since the ER is a site for folding and oligomerization of nascent proteins (1Galili G. Sengupta-Gopalan C. Ceriotti A Plant Mol. Biol. 1998; 38: 1-29Crossref PubMed Google Scholar, 2Okita T.W. Rogers J.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 327-350Crossref PubMed Scopus (141) Google Scholar, 3Vital A. Denecke J. Plant Cell. 1999; 11: 615-628PubMed Google Scholar). However, MCP was detected as the mature enzyme in a microsome preparation in which SH-EP was presented as the proenzyme (Fig. 8, A and B), suggesting the enrichment of the ER in the microsomal preparation, and supporting the possibility that mature MCP is localized in the ER. Most papain-type proteases are synthesized as zymogens with the large N-terminal proregion which is essential for correct folding of the enzyme (32Vernet T. Berti P.J. deMontigny C. Musil R. Tessier D.C. Menard R. Magny M.C. Storer A.C. Thomas D.Y. J. Biol. Chem. 1995; 270: 10838-10846Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), contains intracellular transport signal (33Chrispeels M.J. Raikhel N.V. Cell. 1992; 68: 613-618Abstract Full Text PDF PubMed Scopus (147) Google Scholar), and plays a role in inactivation of the protein (34James M.N.G. Sielecki A.R. Nature. 1986; 319: 33-38Crossref PubMed Scopus (240) Google Scholar, 35Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar, 36Carmona E. Dufour E. Plouffe C. Takabe S. Manson P. Mort J.S. Menard R. Biochemistry. 1996; 35: 8149-8157Crossref PubMed Scopus (189) Google Scholar). The activation mechanisms of the proform of papain-type proteases have been elucidated mainly for propapain (32Vernet T. Berti P.J. deMontigny C. Musil R. Tessier D.C. Menard R. Magny M.C. Storer A.C. Thomas D.Y. J. Biol. Chem. 1995; 270: 10838-10846Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 35Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar), procathepsin L (37Smith S.M. Gottesman M.M. J. Biol. Chem. 1989; 264: 20487-20495Abstract Full Text PDF PubMed Google Scholar, 38Nomura T. Fujisawa Y. Biochem. Biophys. Res. Commun. 1997; 230: 143-146Crossref PubMed Scopus (28) Google Scholar), and procathepsin B (39Mach L. Mort J.S. Glössl J. J. Biol. Chem. 1994; 269: 13030-13035Abstract Full Text PDF PubMed Google Scholar), and the mechanisms are largely conserved among these proenzymes. The N-terminal proregion occluding the active site with antiparallel peptide chains is removed by autocatalytic proteolysis triggered by acidification (40Coulombe R. Grochulaki P. Sivaraman J. Menard R. Mort J.S. Cygler M. EMBO J. 1996; 15: 5492-5503Crossref PubMed Scopus (330) Google Scholar, 41Jerala R. Zerovnik E. Kidric J. Turk V. J. Biol. Chem. 1998; 273: 11498-11504Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). We have also succeeded in in vitroactivation of recombinant pro-SH-EP (22Okamoto T. Yuki A. Mitsuhashi N. Minamikawa T. Eur. J. Biochem. 1999; 264: 223-232Crossref PubMed Scopus (33) Google Scholar), but recombinant pro-MCP was not converted to the mature enzyme under the conditions of acidic and nutral pH (data not shown). An activation mechanism different from that of pro-SH-EP, procathepsin L, and propapain may account for the activation of pro-MCP. In fact, the in vitro activation mechanism of proforms of proteases belonging to the same subfamily as MCP has not yet been resolved. Therefore, the processing profiles of MCP was analyzed by heterologous expression system in insect Sf9 cells. It is notable that mature MCP was detected only in the membrane protein fraction prepared from Sf9 cells expressing MCP, and that the other MCP-related polypeptides of 35, 37, and 38 kDa were detected only in the soluble protein fraction (Fig. 9 A). In addition, MCP activity derived from expressed MCP was detected in the membrane protein fraction (Fig. 9 C). This suggests that the membrane association of pro-MCP in Sf9 cells is needed for correct processing to the mature form, and pro-MCP in the soluble protein fraction is processed abnormally or degraded to the 35-, 37-, and 38-kDa polypeptides. Membrane association may have a crucial role in post-translational processing of pro-MCP.A hydropathy plot for the MCP protein did not show the existence of a hydrophobic transmembrane domain on the MCP polypeptide (data not shown). Membrane association of papain-type proteases has been reported in procathepsin L (42McIntyre G.F. Erikson A.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10588-10592Crossref PubMed Scopus (48) Google Scholar, 43McIntyre G.F. Godbold G.D. Erikson A.H. J. Biol. Chem. 1994; 269: 567-572Abstract Full Text PDF PubMed Google Scholar) and the soybean cysteine protease P34 (44Herman E.M. Melroy D.L. Buckhout T.J. Plant Physiol. 1990; 94: 341-349Crossref PubMed Scopus (38) Google Scholar, 45Kalinski A.J. Weisemann J.M. Matthews B.F. Herman E.M. J. Biol. Chem. 1990; 265: 13843-13848Abstract Full Text PDF PubMed Google Scholar, 46Kalinski A.J. Melroy D.L. Dwivedi R.S. Herman E.M. J. Biol. Chem. 1992; 267: 12068-12076Abstract Full Text PDF PubMed Google Scholar). Procathepsin L associates with microsomal membranes at pH 5 via a 6-amino acid residue region in the N-terminal prosequence, which is similar to the vacuolar targeting sequence of yeast proenzymes, and the binding is essential for transport of the proenzyme from the Golgi complex to lysosomes through a mannose 6-phosphate-independent pathway (43McIntyre G.F. Godbold G.D. Erikson A.H. J. Biol. Chem. 1994; 269: 567-572Abstract Full Text PDF PubMed Google Scholar). However, MCP must associate with the membrane through part of the mature enzyme, rather than the proregion, since mature MCP was detected in the membrane protein fraction (Figs. 8 A and9 A), suggesting that the membrane association process for MCP differs from that for procathepsin L. A candidate amino acid sequence of MCP for association with the microsomal membrane is the N-terminal region of mature MCP, LPANAQKAPILP (Fig. 4 B). The region is positioned upsteam from the N-terminal amino acid residues of the papain-type proteases belonging to the other subfamily, is strongly conserved only among the proteases of the MCP subfamily, and shows high hydrophobicity. This may suggest that this amino acid sequence has some functional role, and investigations to see the biological function of the sequence are now underway in our laboratory. P34, a papain-type protease from soybeans, was first identified as a membrane-associated protease of oil bodies of cotyledon cells of soybean seeds (44Herman E.M. Melroy D.L. Buckhout T.J. Plant Physiol. 1990; 94: 341-349Crossref PubMed Scopus (38) Google Scholar, 45Kalinski A.J. Weisemann J.M. Matthews B.F. Herman E.M. J. Biol. Chem. 1990; 265: 13843-13848Abstract Full Text PDF PubMed Google Scholar), but later reports indicated that p34 is localized in vacuoles, and that disruption of the cells resulted in the release of P34 from vacuoles and subsequently P34 associates with the membranes of oil bodies (46Kalinski A.J. Melroy D.L. Dwivedi R.S. Herman E.M. J. Biol. Chem. 1992; 267: 12068-12076Abstract Full Text PDF PubMed Google Scholar). It was suggested that the association of P34 might be only a fortitous event or that the association reflects some aspect of the functions of P34 (46Kalinski A.J. Melroy D.L. Dwivedi R.S. Herman E.M. J. Biol. Chem. 1992; 267: 12068-12076Abstract Full Text PDF PubMed Google Scholar). The possibility of a merely fortitous association of MCP with the membrane will be ruled out by experiments involving proteinase K treatment of microsomes and subsequent immunoblotting (Fig.8 C). The results strongly supported the conclusion that MCP detected as a membrane-associated protein is localized in the luminal side of the microsomes, and that MCP does not simply bind nonspecifically to the microsomal membrane on the cytoplasmic side during preparation of the microsomes. The specific association of MCP with the luminal membrane is also indirectly supported by the results of immunocytochemistry showing that MCP is localized along the membrane of the ER (Fig. 6 B) and on peripheral regions and membranous structures of specific areas of PSV where degradation of storage proteins is progressing (Fig. 7 B). In addition, the heterologous expression of MCP in Sf9 cells and subsequent sublocalization within the cells revealed that the MCP protein itself possesses the property of associating with the membrane (Fig. 9). From these observations, the features of membrane association of MCP are considered to reflect some biological role. In the ER lumen, soluble proteins are able to move freely, but the movement of membrane-associated proteins is thought to be restricted and it is considered likely that membrane association will result in compartmentalization of the proteins in some region of the lumen. Molecular chaperones in the ER bind to nascent proteins until the nascent proteins are correctly folded or oligomerized (1Galili G. Sengupta-Gopalan C. Ceriotti A Plant Mol. Biol. 1998; 38: 1-29Crossref PubMed Google Scholar, 2Okita T.W. Rogers J.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 327-350Crossref PubMed Scopus (141) Google Scholar, 3Vital A. Denecke J. Plant Cell. 1999; 11: 615-628PubMed Google Scholar). Membrane association of MCP on the luminal side of the ER may reduce the opportunity for assembly with nascent proteins in the lumen of the ER prior to binding of chaperones to the proteins, resulting in the protection of MCP against nonspecific proteolysis of the protein. Although the in vivo substrates of MCP in the ER lumen remain to be identified, at least MCP was revealed to process the C-terminal prosegment of SH-EP in vitro. One hypothesis is that MCP in the ER lumen is involved in cleavage of the C-terminal region of reticuloplasmins possessing K(H)DEL-tails, resulting in escape from the ERD2 retention system and degradation of the proteins for turnover processes in the vacuoles. We herein postulate that MCP is localized in both the ER and PSV as a membrane-associated protease, and plays dual roles in cells, in the post-translational cleavage during the C-terminal processing of pro-SH-EP, and in the degradation of storage proteins in PSV. The endoplasmic reticulum (ER)1 is the port of entry of proteins into the endomembrane system. In this organelle, there are a number of soluble proteins, membrane proteins, and molecular chaperones which are involved in folding, glycosylation, assembly, and maturation of nascent proteins (1Galili G. Sengupta-Gopalan C. Ceriotti A Plant Mol. Biol. 1998; 38: 1-29Crossref PubMed Google Scholar, 2Okita T.W. Rogers J.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 327-350Crossref PubMed Scopus (141) Google Scholar, 3Vital A. Denecke J. Plant Cell. 1999; 11: 615-628PubMed Google Scholar). The soluble proteins localized in the lumen of the ER have a retention signal, KDEL or HDEL, at the C terminus (4Munro S. Pelham H.B.R. Cell. 1987; 48: 899-907Abstract Full Text PDF PubMed Scopus (1562) Google Scholar, 5Pelham H.B.R. Annu. Rev. Cell Biol. 1989; 5: 1-23Crossref PubMed Scopus (539) Google Scholar, 6Napier R.M. Fowke L.C. Hawes C.R. Lewis M. Pelham H.B.R. J. Cell Sci. 1992; 102: 261-271Crossref PubMed Google Scholar), and this signal is known to be recognized by the K(H)DEL receptor on the Golgi complex, which mediates retrieving K(H)DEL-tailed proteins to the ER. The molecular mechanisms of the ER retention of soluble proteins are conserved through animal, plant, and yeast cells (7Lee H.I. Gal S. Newman T.C. Raikhel N.V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11433-11437Crossref PubMed Scopus (91) Google Scholar, 8Denecke J. De Rycke R. Botterman J. EMBO J. 1992; 11: 2345-2355Crossref PubMed Scopus (231) Google Scholar, 9Herman E.M. Tague B.W. Hoffman L.M. Kjemtrup S.E. Chrispeels M.J. Planta. 1990; 182: 305-312Crossref PubMed Scopus (69) Google Scholar). In several kinds of plants, unique papain-type proteinases possessing a C-terminal KDEL sequence have been identified (10Akasofu H. Yamauchi D. Mitsuhashi W. Minamikawa T. Nucleic Acids Res. 1989; 17: 6733Crossref PubMed Scopus (59) Google Scholar, 11Becker C. Shutov A.D. Nong V.H. Senyuk V.I. Jung R. Horstmann C. Fischer J. Nielsen N.C. Müntz K. Eur. J. Biochem. 1995; 228: 456-462Crossref PubMed Scopus (82) Google Scholar, 12Guerrero C. de la Calle M. Reid M.S. Valpuesta V. Plant Mol. Biol. 1998; 36: 565-571Crossref PubMed Scopus (102) Google Scholar, 13Tanaka T. Yamauchi D. Minamikawa T. Plant Mol. Biol. 1991; 16: 1083-1084Crossref PubMed Scopus (47) Google Scholar, 14Valpuesta V. Lange N.E. Guerrero C. Reid M.S. Plant Mol. Biol. 1995; 28: 575-582Crossref PubMed Scopus (109) Google Scholar, 15Schmid M. Simpson D. Kalousek F. Gietl C. Planta. 1998; 206: 466-475Crossref PubMed Scopus (77) Google Scholar, 16Cercos M. Santamaria S. Carbonell J. Plant Physiol. 1999; 119: 1341-1348Crossref PubMed Scopus (53) Google Scholar). One such KDEL-tailed cysteine protease, designated SH-EP, was first isolated from cotyledons of germinated Vigna mungo seeds as the enzyme responsible for degradation of storage proteins accumulated in protein storage vacuoles (PSV) of cotyledon cells (10Akasofu H. Yamauchi D. Mitsuhashi W. Minamikawa T. Nucleic Acids Res. 1989; 17: 6733Crossref PubMed Scopus (59) Google Scholar, 17Mitsuhashi W. Koshiba T. Minamikawa T. Plant Physiol. 1986; 80: 628-634Crossref PubMed Google Scholar). SH-EP is synthesized on membrane-bound ribosomes as a 43-kDa precursor through co-translational cleavage of the signal peptide, and the precursor is processed to the 33-kDa mature enzyme via 39- and 36-kDa intermediates during or after transport to the vacuoles (18Mitsuhashi W. Minamikawa T. Plant Physiol. 1989; 89: 274-279Crossref PubMed Scopus (65) Google Scholar, 19Okamoto T. Nakayama H. Seta K. Isobe T. Minamikawa T. FEBS Lett. 1994; 351: 31-34Crossref PubMed Scopus (33) Google Scholar). The function of the KDEL-tail on a cysteine protease whose final destination is the PSV has been an interesting question. Based on analysis of the heterologous expression of SH-EP and a KDEL deletion mutant of SH-EP in insect Sf9 cells and subcellular fractionation of cotyledon cells, it was proposed that the KDEL-tail of SH-EP functions to store the enzyme as a transient zymogen in the ER, and that the conversion of the 43-kDa SH-EP into the 42-kDa form in/at the ER is accompanied by the removal of the C-terminal propeptide containing the KDEL-tail (20Okamoto T. Minamikawa T. Edward C. Vakharia V. Herman E. J. Biol. Chem. 1999; 274: 11390-11398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). From these observations, the protease responsible for removal of the C-terminal propeptide of SH-EP has been supposed to exist in the lumen of the ER (20Okamoto T. Minamikawa T. Edward C. Vakharia V. Herman E. J. Biol. Chem. 1999; 274: 11390-11398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Recently, Toyooka et al. (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar) showed that in cotyledon cells of V. mungo seedlings, a proform of SH-EP synthesized in the ER accumulated at the edge or middle regions of the ER where the transport vesicle was formed. The vesicle, containing a large amount of pro-SH-EP, termed KV, budded off from the ER, bypassed the Golgi complex, and fused to PSV (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar). It was proposed that the KDEL-tail of SH-EP functions as the signal for accumulation of pro-SH-EP at the edge or middle regions of the ER where formation of KV proceeds (21Toyooka K. Okamoto T. Minamikawa T. J. Cell Biol. 2000; 248: 453-463Crossref Scopus (151) Google Scholar). In this study, the protease involved in the processing of 43-kDa SH-EP (KDEL-attached form) into 42-kDa SH-EP (KDEL-removed form) was purified to homogeneity from microsomes of cotyledons of V. mungoseedlings, and a cDNA clone for the enzyme was isolated. Immunocytochemical and sucrose gradient analyses of cotyledon cells were carried out to determine the intracellular localization of the protease. The protease was also expressed in insect Sf9 cells in order to observe the characteristics of the membrane association of the enzyme. The possible dual functions of this enzyme in cells will be discussed. DISCUSSIONAlthough MCP was initially isolated as a protease which is involved in post-translational cleavage of the C-terminal propeptide of SH-EP containing the KDEL-tail within the ER, immunocytochemical analysis of cotyledon cells revealed that MCP is localized in PSV as well as the ER, and the amino acid sequence of MCP deduced from its cDNA indicated that the enzyme is a member of papain family which are potentially degradative enzymes. The involvement of MCP in degradation of storage proteins is consistent with the report that an MCP homologue from Vicia faba was isolated as an enzyme responsible for globulin hydrolysis in cotyledons of germinated V. faba seeds (30Yu W.J. Greenwood J.S. J. Exp. Bot. 1994; 271: 261-268Crossref Scopus (25) Google Scholar, 31Yu W.J. Greenwood J.S. Plant Physiol. PGR. 1996; 112: 862Google Scholar). It has been shown that there are some kinds of proteinases in the cotyledons of germinated V. mungo seeds (17Mitsuhashi W. Koshiba T. Minamikawa T. Plant Physiol. 1986; 80: 628-634Crossref PubMed Google Scholar, 28Okamoto T. Minamikawa T. Plant Mol. Biol. 1999; 39: 63-73Crossref PubMed Scopus (51) Google Scholar) and other papain-type proteinases are thought to exist in the cotyledons, however, only MCP showed the proteolytic activity that converts the recombinant 43-kDa SH-EP (C152G) to the 42-kDa form. In addition, general cysteine proteinase, papain, cleaved the recombinant 43-kDa SH-EP (C152G) to the 33-kDa form through removal of the N-terminal prosequence of the proenzyme. This processing profile is apparently different from that of the recombinant 43-kDa SH-EP (C152G) by MCP. This suggest the possibility that the processing activity for the conversion of the recombinant 43-kDa SH-EP (C152G) to the 42-kDa form is unique to MCP.The present results of sucrose gradient centrifugation and immunocytochemistry of cotyledon cells suggest the possibility that MCP exists as an active mature enzyme in the ER in association with the membrane, although papain-type proteases have not been expected to be localized in the ER lumen since the ER is a site for folding and oligomerization of nascent proteins (1Galili G. Sengupta-Gopalan C. Ceriotti A Plant Mol. Biol. 1998; 38: 1-29Crossref PubMed Google Scholar, 2Okita T.W. Rogers J.C. Annu. Rev
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