Seeds of the common bean (Pbaseolus vulgaris) contain a plant defense protein that inhibits the a-amylases of mammals and insects. This a-amylase inhibitor (aAl) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M,) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Pbaseolus, we found that antibodies to uAl recognize large (M. 30,000-35,000) polypeptides as well as typical aAl processing products (M, 15,000-18,000). aAl activity was found in all extracts that had the typical aAl processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant aAl in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-aAl when the gene is expressed in tobacco. When pro-aAl was separated from mature aAl by gel filtration, pro-aAl was found not to have a-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.Many secretory proteins in plant, mammalian, and funga1 cells are synthesized as preproproteins that undergo cotranslational removal of the signal peptide, as well as posttranslational proteolytic maturation processing (Neurath, 1986). Such maturation processing, which may occur as a singlestep or a multistep process, converts an inactive proprotein into an active mature protein through the release of a confonnational constraint within the proprotein. Examples are the release of peptide hormones and growth factors in mammalian and yeast cells, the activation of enzyme zymogens, especially proteases, the complement system, and blood coagulation. Processing is often accompanied by the loss of one or more polypeptide domains that are present in the proprotein but absent from the mature protein. Limited proteolysis requires that enzyme and substrate be present in the same compartment and that the processing enzyme be specific for the cleavage and selective for the substrate.
A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretary pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins.The secretary system of plant cells delivers proteins to the vacuole, the tonoplast, the plasma membrane, and the cell wall/extracellular space. In addition, proteins that enter the secretary system may be retained in the endoplasmic reticulum or in various compartments of the Golgi complex. The first step common to the transport of all these proteins is translocation across the ER membrane (25). Once
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