Leonard Beevers

Molecular Cloning and Further Characterization of a Probable Plant Vacuolar Sorting Receptor

BP-80 is a type I integral membrane protein abundant in pea (Pisum sativum) clathrin-coated vesicles (CCVs) that binds with high affinity to vacuole-targeting determinants containing asparagine-proline-isoleucine-arginine. Here we present results from cDNA cloning and studies of its intracellular localization. Its sequence and sequences of homologs from Arabidopsis, rice (Oryza sativa), and maize (Zea mays) define a novel family of proteins unique to plants that is highly conserved in both monocotyledons and dicotyledons. The BP-80 protein is present in dilated ends of Golgi cisternae and in “prevacuoles,” which are small vacuoles separate from but capable of fusing with lytic vacuoles. Its cytoplasmic tail contains a Tyr-X-X-hydrophobic residue motif associated with transmembrane proteins incorporated into CCVs. When transiently expressed in tobacco (Nicotiana tabacum) suspension-culture protoplasts, a truncated form lacking transmembrane and cytoplasmic domains was secreted. These results, coupled with previous studies of ligand-binding specificity and pH dependence, strongly support our hypothesis that BP-80 is a vacuolar sorting receptor that trafficks in CCVs between Golgi and a newly described prevacuolar compartment.

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (lpomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.

Structural Requirements for Ligand Binding by a Probable Plant Vacuolar Sorting Receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain–EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.

The development of proteolytic activity and protein degradation during the germination of Pisum sativum L.

SummaryThe change in protein content and composition of the cotyledons of Pisum sativum L. cv. Burpeeana during germination was studied. Protein depletion from the cotyledons was slow during the first 4 days of germination but became rapid on the 5th day and by the 16th day the majority of the protein had disappeared. During the first 4 days the depletion of the globulins exceeded that of the albumins; legumin appeared to be degraded slightly more rapidly than vicilin during the early phase of germination. Sodium-dodecylsulfate (SDS) electrophoresis of SDS- and dithiothreitol-dissociated globulins indicated that before rapid protein depletion there were marked changes in the component composition of the major globulins legumin and vicilin. The onset of rapid protein depletion was associated with an increase in the level of an acid-sulfhydryl protease in the cotyledons. These findings indicate that the reserve globulins undergo modifications prior to their eventual hydrolysis.

Nitrogen Metabolism in Plants

Although plants or plant parts can be cultured on various organic nitrogen forms, particularly amino acids, under natural conditions the majority of plants depend upon inorganic nitrogen sources. Exceptions to this may be various parasitic and semi-parasitic plants which may be provided with organic nitrogenous nutrients of host origin, and insectivorous plants which partially satisfy their nitrogen demands by utilizing organic nitrogen constituents released by lysis of trapped insects.

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.

Clathrin-Coated Vesicles in Plants

This chapter focuses on current knowledge of coated vesicles from plant systems. It is apparent that although the studies have been directed by the much greater volume of information from animal systems, there has been considerable progress in our understanding of the function and biochemical characterization of clathrin-coated vesicles from plants over the past decade. Ultrastructural studies have demonstrated coated vesicles in a variety of plant cells. Within the cells, the vesicles are involved in endocytosis, membrane recycling, and the intracellular transport of vacuolar proteins. Improved isolation procedures have facilitated the biochemical characterization of clathrin-coated vesicles. To date, coat components of 180-kDa clathrin have been identified; however, the identity of light chains remains enigmatic. Adaptor peptides have been isolated and potential receptor proteins for vacuolar targeted proteins identified. The functioning of coated vesicles requires removal of the clathrin coat and appropriate uncoating ATPase has been identified. The dissociation of the receptor(s) and targeting ligands of the transported protein appears to involve a proton-pumping vacuolar H+-ATPase associated with the vesicle. It is suggested that the capacity to routinely isolate vesicles, combined with techniques of molecular biology, should lead to a more rapid accumulation of information on the function and biochemistry of clathrin-coated vesicles from plants.

Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies.

Crude protein extracts from leaves of pea (Pisum sativum L.) labeled with an L-14C-amino-acid mixture or [35S]methionine, were treated with antibodies prepared against nitrite reductase (NiR; EC 1.6.6.4). When the immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide with the same mobility as that of the native NiR was detected. By using darkened or illuminated leaves in the absence or presence of nitrate, it has been confirmed that nitrate is required in the in-vivo synthesis of NiR and that this synthesis is stimulated by light. Cell-free translation with a wheat-germ extract primed with polysomes from illuminated leaves treated with nitrate yielded polypeptides of a wide range of molecular weights (Mrs). Two polypeptides were immunoprecipitated from the translational products by anti-NiR serum. The mobility of one of them on SDS-PAGE corresponded to that of NiR while the other had a slightly higher Mr. It is concluded that NiR is synthesized as a heavy-molecular-weight precursor. Nitrate appears to regulate NiR synthesis by triggering transcription whereas the light may control the level of transcription or translation.

The influence of methionine sulfoximine on photosynthesis and nitrogen metabolism in excised pepper (Capsicum annuum L.) leaves

Abstract The influence of methionine sulfoximine (MSO) on the photosynthesis and NH 4 + accumulation in excised pepper ( Capsicum annuum L.) leaves has been studied at 2 and 20% oxygen levels. Petiolar feeding with 10 mM MSO decreased the photosynthetic rate by 86% in 2 h when 20% O 2 was provided. During this 2-h period, the endogenous NH 4 + level rose from 0.9 to 11.0 μmol/g fresh wt. In the presence of 2% O 2 , MSO treated leaves showed a decline of only 18% in photosynthesis which was accompanied by a rise in NH 4 + level from 0.9 to 2.65 μmol/g fresh wt. In repeated experiments a negative correlation was obtained between the endogenous level of NH 4 + and photosynthesis ( r = 0.89). Pronounced accumulation of NH 4 + in MSO treated leaves occured only when conditions favored photorespiration.

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [14C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.