Degradation of abnormal proteins in growing yeast

Abstract Degradation of abnormal proteins in Saccharomyces cerevisiae , synthesized in an ethanol medium in the presence of 0.1 mM canavanine, proceeded more rapidly than that of the normal ones, the degradation constant of the 'long-lived' fraction being most affected. Higher concentrations of the analogue had an adverse effect on protein degradation during subsequent growth. Degradation of normal and abnormal proteins was suppressed by glucose but was increased substantially in cell-free extracts. This suggests that their internalization into the digestive organelle may be an important step in protein catabolism in yeast.     

Effects of canavanine on IAA- and fusicoccin-stimulated cell enlargement, proton extrusion and potassium uptake in maize coleoptiles

In maize coleoptiles (Zea mays F₁ XL 640A, cv. Dekalb) canavanine and cycloheximide strongly and simultaneously inhibit cell elongation, H⁺ extrusion and K⁺ uptake, induced by IAA. They inhibit also, although to a much lesser degree, the same phenomena induced by fusicoccin. Cycloheximide severely depresses the incorporation of leucine into proteins, while canavanine leaves leucine incorporation almost unchanged. The data confirm that elongation, H⁺ extrusion and K⁺ uptake can be regarded as correlated processes; they also support the view that normal protein synthesis is essential for IAA-induced growth, while this requirement is only partial in growth stimulated by fusicoccin.     

The Effects of Canavanine on Protein and Nucleic Acid Synthesis in Canavanine Resistant and Sensitive Species of Higher Plants

The effectiveness of the inhibitor, canavanine, was evaluated by examining its action in Canavalia ensiformis and Glycine max. Isolated roots were grown in culture tubes containing White's medium plus canavanine or arginine. A differential effect of canavanine on the incorporation of precursors of DNA, RNA, and protein was found, which is assumed to be related to the ability of the plant to utilize canavanine in reactions typically involving arginine. Canavanine was not found to affect DNA, RNA, or protein synthesis in Canavalia ensiformis, a plant in which this amino acid is synthesized naturally. In the canavanine sensitive species, Glycine max, of the same subfamily Papilionoideae, canavanine was observed to inhibit strongly DNA, RNA, and protein synthesis. A primary inhibition of the RNA synthesizing system is suggested. The data indicate the canavanine inhibitions are more complex than a simple competition with arginine in protein synthesis.     

Effects of Canavanine on Arginine Utilization in Plants with Special Reference to Mitotic Activity

Excised primary roots of Phaseolus vulgaris L. were treated with canavanine and the effect on arginine utilization was studied. Arginine utilization was observed to be depressed to a low level by canavanine (1.8 × 10^?4M) in rapidly dividing and growing tissues. In addition, canavanine inhibited arginine utilization to a lesser degree in root sections composed of mature and non-dividing tissues. This demonstrated that canavanine inhibition is not limited to those tissues where active DNA synthesis occurs. Canavanine was observed to inhibit the onset of mitosis in primary roots as evidenced by a significant reduction of the frequency of mitotic figures. In addition, this amino acid was found to affect the course of mitosis once it was initiated by altering the relative frequency of mitotic stages, decreasing the percentage of prophase figures and increasing the percentage observed in the telophase. An influence on the rate of spiralization and despiralization of the chromosomes is suggested. The effects on mitosis are probably related to growth-inhibitory and toxic effects of canavanine. The possible function of canavine as an allelopathic substance is hypothesized.     

Canavanine distribution in jackbean fruit during fruit growth

Summary Canavanine is an arginine analogue found in the seeds of many common legumes and is known to inhibit protein synthesis and growth in a number of organisms. Yet canavanine may comprise as much as 4% of the seed dry weight of the jackbean (Canavalia ensiformis).Canavanine is accumulated during earlier development in the pod, but disappears upon ripening. A corresponding increase in seed canavanine of about the same magnitude as the loss in the pod takes place during this latter time, but there is a subsequent significant increase of canavanine content of the seed after all detectable canavanine has disappeared from the pod. The first of these changes suggests synthesis of canavanine in the pod and transport into the seeds while the second one indicates a synthesis of canavanine in the seeds themselves, or possibly in the leaf or pod with rapid translocation to the seed.Canavanine was found to be at its highest concentration in the seed coats and pods when they were growing most rapidly and to gradually decline afterwards; however, the canavanine concentration of the seeds was found to be constant throughout fruit development.The pattern of canavanine mobilization in jackbean fruits was quite similar to the known pattern of total nitrogen mobilization typical of other leguminous fruits. This is consistent with a role as a nitrogen transport and storage compound.University of Tennessee, Department of Botany, Contribution N. Ser. No. 279.     

The Effect of Some Protein and Non-Protein Amino Acids on the Growth of Cladosporium herbarum and Trichothecium roseum

The effect of ten amino acids as the sole nitrogen source for the growth of Cladosporium herbarum (Link.) Fr. and Trichothecium roseum (Bull.) Link. was studied in order to clarify the fungus-host plant relationship. Special attention was paid to some rare non-protein amino acids of legumes. The best nitrogen sources for both fungi were γ-aminobutyric acid, arginine, serine and proline. Cladosporium could use homoarginine and canavanine, but these two amino acids were not used by Trichothecium when each was given as the only nitrogen source. Both fungi utilized ornithine, homoserine and a,γ-diaminobutyric acid to a limited extent. Pipecolic acid was not growth promoting. The growth-retarding effects of rare non-protein amino acids (homoarginine, canavanine, a,γ-diaminobutyric acid and pipecolic acid) were usually reversed by higher concentrations of their normal analogues. It is possible that rare non-protein amino acids may slightly protect the host plant against fungal infections, but there are clear differences between fungi in their reaction to non-protein amino acids.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

l-Canavanine: a higher plant insecticidal allelochemical

Summary. l-Canavanine, l-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease. The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins. The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical. Received February 1, 2000 Accepted June 4, 2000     

Effect of Canavanine on Murine Retrovirus Polypeptide Formation

Canavanine is an arginine analog which is widely used to inhibit proteolytic processing of viral polyproteins. Certain results obtained with canavanine have suggested that it may have other effects. Therefore, we examined the effects of canavanine on the cell-free synthesis of murine retrovirus proteins. It was found that the electrophoretic mobility of the major gag-related cell-free product of both Rauscher murine leukemia virus (R-MuLV) and Moloney murine sarcoma virus 124 (Mo-MuSV-124) RNA was dependent on the concentration of canavanine used during translation. As the canavanine concentration was increased up to 4 mM, the apparent size of the major gag-related polypeptide also increased from 65,000 (R-MuLV RNA) or 63,000 (Mo-MuSV-124 RNA) to approximately 80,000 daltons. Additional increases in the canavanine concentration up to 12 mM did not increase the size of the gag gene product beyond 80,000 daltons. This change in electrophoretic mobility appeared to be due to a substitution of canavanine for arginine residues in the polypeptides, not to a change in their actual size. If amber suppressor tRNA and canavanine were used together during translation of Mo-MuSV-124 RNA and Mo-MuLV RNA, the results were also in agreement with this proposal. Translation experiments done with ovalbumin mRNA and mengovirus 35S RNA indicated that canavanine incorporation caused a shift in the electrophoretic mobility of ovalbumin from 43,000 to 45,000 daltons and caused the appearance of two slightly larger polypeptides in the 155,000- and 115,000- dalton regions of the mengovirus RNA cell-free product.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P₁₁₀, P₈₈, P_71,72, and P₂₃ had unique peptide maps. Evidence of a 27,000 dalton precursor to P₂₃ was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P₈₈ and P_71,72 increased from basal to new plateau levels in about 1.5 hours, while P₂₃ required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the metabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.     

Initial kinetics of Protein turnover in growing yeast

Proteins in yeast growing in a medium with glucose or ethanol as carbon source were pulse-labelled by a 20-min incubation with¹⁴C-leucine. The proteins in cells labelled and growing in a glucose medium were stable; when this population was transferred to the ethanol medium, the proteins were degraded at a rate of 1.1 %/h. The population labelled and growing in an ethanol medium displayed a fraction of short-lived proteins (about 4 %), decaying with a half-life of 0.5 h. The size of the short-lived protein fraction increased slightly after shifts to a glucose as well as to a starvation medium. The residual long-lived proteins underwent a turnover of 1.3 –1.4 %/h in the ethanol or the starvation medium and of 0.3 %/h in the glucose medium, respectively. Proteins labelled in the presence of canavanine or ethionine were degraded at only a slightly greater rate than the normal proteins. Participant of the UNESCO Postgraduate Course “On Modern Problems in Biology”.     

The effect of L-canavanine and L-canaline on the hemolymph amino acid composition of the tobacco hornworm,Manduca sexta (L.) (Sphingidae)

Tobacco hornworm larvae, Manduca sexta (L.) (Sphingidae), were administered L-canaline either by parenteral injection or by dietary consumption. The overt toxicity and the alteration of hemolymph amino acids caused by these nonprotein amino acids were evaluated. The LD₅₀ value for parenterally administered canavanine and canaline is 1.0 and 2.5 mg/g fresh body weight, respectively. A dietary concentration of 5.2 mM for canavanine and over 20 mM for canaline represent the respective LC₅₀ values. A large percentage of the larvae reared on diets supplemented with additional arginine, ornithine, or 2,4-diaminobutyric acid in addition to canavanine or canaline were unable to complete larval-pupal ecdysis. These toxic effects were associated with a decreased glutamic acid hemolymph titer and dramatically elevated ornithine. On the other hand, larvae administered canavanine or canaline alone, either by dietary consumption or parenteral injection, experienced less drastic developmental aberrations. These symptoms were in some cases correlated with increased ornithine and glutamic acid titers. Evidence is presented that even a canavanine- and canaline-sensitive insect such as M. sexta has a marked ability to eliminate these protective allelochemicals.     

Monophyletic subgroups of the tribe Millettieae (Leguminosae) as revealed by phytochrome nucleotide sequence data

Phylogenetic analysis of phytochrome (PHY) genes reveals the identity and relationships of four PHY loci among papilionoid Leguminosae. A phylogenetic analysis of loci combined according to species suggests that most of the tribe Millettieae belongs to one of two monophyletic clades: the Derris–Lonchocarpus or the Tephrosia clade. Together these two form a monophyletic group that is sister to a lineage represented by Millettia grandis of Millettia sect. Compresso-gemmatae. Collectively, this large monophyletic group is referred to as the Millettieae-core group, which based on our sampling, includes species of Millettieae that do not accumulate the nonprotein amino acid canavanine and that mostly have pseudoracemose or pseudopaniculate inflorescences. This new phylogenetic framework assists in targeting additional taxa for future sampling. For example, the “American Derris” (Deguelia), which accumulate canavanine, might not be members of the Millettieae core group. Afgekia is also predicted not to be a member because it accumulates canavanine and has an inflorescence of terminal racemes. PHY gene analysis specifically reveals that certain genera traditionally classified in Millettieae are actually distantly related to the Millettieae core group, such as Austrosteensia, Callerya, Craibia, Cyclolobium, Fordia, Platycyamus, Poecilanthe, and Wisteria.     

The occurrence of canavanine in seeds of the tribe robinieae

Fifty-five species representing 14 genera of Robinieae are reported to accumulate canavanine in seed. This represents approximately one-third the number of species and two-thirds the number of genera in the tribe and indicate Robinieae to be the only tribe with a predominantly New World distribution to accumulate canavanine. The only member of Robinieaea found not to accumulate canavanine is the genus Diphysa. The lack of canavanine in seed of this genus lends systematic weight to characters shared in common by Diphysa with the tribe Aeschynomeneae, and a re-evaluation of the tribal position of Diphysa may be in order.     

Effects of canavanine on the secretion of plasma proteins by Hep G2 cells

Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins.     

Structural aberrations in T-even bacteriophage. VII. In vitro analysis of the canavanine-mediated inhibition of proteolytic cleavage.

Canavanine arrests a critical function in head morphogenesis and the potential for forming giant T-even phage particles termed lollipops is induced. Formation of the particles requires the addition of arginine and the restoration of normal functions. We now report on an investigation into the effects of canavanine on both the T4-induced proteolytic activity and on the substrate proteins. Using an in vitro cleavage assay we have shown that the gene 21-dependent proteolytic activity from canavanine-treated extracts is markedly inhibited, whereas the substrate proteins retain a high susceptibility for cleavage. The proteolytic activity in extracts treated with canavanine followed by arginine is readily detectable, and proteins previously synthesized in the presence of canavanine can be cleaved. Protein synthesis is apparently required for the appearance of the proteolytic activity after the canavanine-arginine treatment. Mixing experiments suggest the requirement for a component of the gene 21-dependent proteolytic activity that is not coded for by gene 21.     

Proteins and polyamines during dormancy breaking of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.