Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds

Abstract

A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis.When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translaffon in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 ± 10C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydrylendopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33 kD mature enzyme.Although it is generally accepted that legume seed proteins are mobilized during germination by a combination ofvarious peptide hydrolases (2, 6, 20, 28), a number of reports have shown that endopeptidases play a greater role in the mobilization process (3,7,8,15,19,24,25). However, the mechanism of regulation of these enzyme activities during legume seed germination is not yet clearly understood.In previous studies (17) zymes responsible for storage protein degradation from V. mungo seedlings. Experiments using a specific antiserum and an in vitro translation system suggest that the V. mungo sulfhydryl-endopeptidase (33 kD) is synthesized on membrane-bound polysomes as a large inactive 45 kD precursor, which is cotranslationally processed to the 43 kD intermediate through the cleavage of a 2 kD signal sequence. After analyzing the results of in vitro processing experiments with enzyme extracts from cotyledons, we postulate that the 43 kD intermediate of sulfhydryl-endopeptidase is processed further to the 33 kD mature enzyme through 39 and 36 kD intermediates, and that the endopeptidase becomes fully active when the 36 kD form is cleaved to the 33 kD mature form. MATERIALS AND METHODS Plant MaterialsVigna mungo seeds were germinated on layers of wet filter paper at 27 ± 10 C in the dark. Cotyledon...

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

“…These proteins are excellent candidates for molecules that play a critical role in tapetum physiological processes. By contrast, the TA56 mRNA encodes a protein similar to the thiol endopeptidases that are present in germinating cotyledons (Mitsuhashi and Minamikawa, 1989) and senescing leaves (Miller and Huffaker, 1982). The appearance of the TA56 mRNA in the connective, stomium, and circular cell cluster just before their degeneration (Figure 6) suggests that the TA56 thiol endopeptidase may aid in the degenerative process by either activating hydrolytic enzymes that degrade cellular macromolecules, or by functioning as a hydrolytic enzyme, or both.…”

“…Seurinck, J. Leemans, and R.B. Goldberg, unpublished results) showed that the TA32136 and TA56157 groups represented mRNAs encoding lipid transfer proteins (Bouillon et al, 1987;Takishima et al, 1988) and thiol endopeptidase proteins (Miller and Huffaker, 1982;Mitsuhashi and Minamikawa, 1989), respectively. In addition, the TA1 31 TA29 group represented mRNAs that encoded glycinerich proteins with properties of cell wall proteins (Condit and Meagher, 1986;Keller et al, 1988;Varner and Cassab, 1988).…”

“…Tobacco plants were transformed with a TA29IDTA gene as described in Methods. The TA29/DTA gene contained a TA29 5' DNA fragment containing nucleotides -1477 to +51 (Mariani et al, 1990;Seurinck et al, 1990) fused with the DTA gene coding sequence (Greenfield et al, 1983;Maxwell et al, 1986;Palmiter et al, 1987). TA13 TA29 TA20 TA25 TA26 TA36 TA39 TA51 TA4 _ .…”

“…By contrast, DNA fragments representing portions of this region were inserted into a similar GUS vector that contained the CaMV 35s gene minimal promoter (nucleotides -52 to +8; Benfey et al, 1990aBenfey et al, , 1990b. The chimeric TA29/DTA gene was constructed exactly as described by Mariani et al (1990) using a 5' TA29 gene fragment containing nucleotides -1 477 to +51 (Figure 1 OA) and the DTA coding sequence (Greenfield et al, 1983;Maxwell et al, 1986;Palmiter et al, 1987).…”

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

“…The enzyme protein and its activity were also detected in cotyledons and other organs of seedlings. EP-Cl was found to be immunologically identical to SH-EP, the major Cys endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (Mitsuhashi et al, 1986;Akasofu et al, 1989;Mitsuhashi and Minamikawa, 1989), although there was a small difference in molecular mass between them. In fact, the amino acid sequence deduced from EP-Cl cDNA, which we reported separately (Tanaka et al, 1991), had a high degree of homology (94%) with that from SH-EP cDNA (Akasofu et al, 1989).…”