In vivo half-life of a protein is a function of its amino-terminal residue.

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

Ubiquitylation in plants: a post-genomic look at a post-translational modification.

In this article, we summarize Arabidopsis genes encoding ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and an additional selected set of proteins related to ubiquitylation. We emphasize comparisons to components from Saccharomyces cerevisiae, with occasional reference to animals. Among the E1 and E2s, Arabidopsis usually has two to four probable orthologs to one yeast gene. Also, Arabidopsis has genes with no likely ortholog in yeast, although they often have potential orthologs in animals. The large number of components with known function in ubiquitylation indicates that this process plays a complex role in cellular physiology.

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Tobacco plants perturbed in the ubiquitin-dependent protein degradation system accumulate callose, salicylic acid, and pathogenesis-related protein 1.

Transgenic tobacco plants expressing an inhibitor of ubiquitin-dependent protein degradation, ubiquitin variant ubR48, spontaneously formed necrotic lesions and displayed altered responsiveness to tobacco mosaic virus attack. These plants were analyzed for the accumulation of defense-related compounds and the expression of pathogenesis-related proteins, which serve as convenient markers for systemic acquired resistance. Callose was detected in the cells of vascular bundles and in the leaf blade. In addition, ubR48 transgenic plants constitutively accumulated enhanced levels of salicylic acid (SA) and/or its glucoside. Accumulation of SA glucoside coincided with high levels of pathogenesis-related protein 1, underscoring the similarity of certain changes in ubR48-expressing plants to an authentic defense reaction.

The degradation signal in a short-lived protein.

Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal. To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a "stabilizing" or a "destabilizing" amino-terminal residue were expressed in the yeast S. cerevisiae and their in vivo half-lives compared. The amino-terminal degradation signal is shown to comprise two distinct determinants. One, discovered previously, is the protein's amino-terminal residue. The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue. The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal.

RNA packaging of yeast retrotransposon Ty1 in the heterologous host, Escherichia coli.

Expression of components of the yeast retrotransposon Ty1 in E. coli was used to study early steps of retrotransposition. We find that polypeptides encompassing the capsid-forming component of Ty1 can assemble into particles in the heterologous host. Ty RNA can be detected in particle fractions. RNA packaging depends on features in the 5' part of Ty RNA, because deletion of 5' proximal sequences leads to decreased packaging efficiency. Protein domains required for the RNA packaging process reside between amino acids 146 and 394 of the capsid protein. The data presented also indicate that several early steps in the Ty1 life cycle can occur in a cellular environment which differs from yeast cytoplasm, supporting the notion that these steps are independent of host factors.

Use of a reporter transgene to generate arabidopsis mutants in ubiquitin-dependent protein degradation.

Ubiquitin-dependent proteolysis is a major proteolytic pathway in the cytoplasm and nucleus of eukaryotic cells. We introduced a gene encoding a substrate for this pathway into the genome of Arabidopsis thaliana. The transgene codes for a hybrid protein consisting of dihydrofolate reductase (DHFR, EC 1.5.1.3) fused to a degradation signal that is specifically recognized by components of the ubiquitin-dependent proteolysis pathway. Elevated concentrations of the DHFR protein confer resistance to the drug methotrexate, but rapid degradation prevents accumulation of the protein in the plant. Therefore, transgenic A. thaliana lines expressing the DHFR fusion protein are methotrexate-sensitive. Selection for mutants resistant to methotrexate resulted in plants impaired in degradation of the DHFR model substrate, as shown by an increase in protein level in the mutants.

Intraspecific length heterogeneity of the rDNA-IGR in Arabidopsis thaliana due to homologous recombination.

A new heterogeneity of the rDNA spacer of Arabidopsis thaliana, resulting from variation in copy number of the so-called 'C' repeat located downstream of the presumptive polymerase I promoter, is reported. Variation is shown to occur within and between ecotypes. PCR analysis and sequence comparison suggests that the observed length heterogeneity is due to homologous recombination.

Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity, but not N-end rule degradation deficiency, of Saccharomyces cerevisiae rad6 mutants.

AtUBC2 of Arabidopsis thaliana encodes a structural homolog of the RAD6 gene of Saccharomyces cerevisiae with approximately 65% identical amino acids. Like structural homologs from other organisms, AtUBC2 lacks the carboxyl-terminal extension of mostly acidic amino acids which is present in Rad6p. AtUBC2 was expressed in S. cerevisiae rad6 mutants. It was found to partially complement the UV sensitivity and reduced growth rate of rad6 mutants at elevated temperatures. AtUBC2 however, has no apparent influence on the degradation of N-end rule substrates in the heterologous host.

Ribosomal transcription units integrated via T-DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana.

Ribosomal repeat units of Arabidopsis thaliana were introduced into the A. thaliana genome via Agrobacterium-mediated transformation. Ribosomal transgenes integrated into chromosomal regions outside the nucleolus organizers. Cytological data suggest that the transgenes associate with a nucleolus. To allow detection of transgenic rRNA, a short extension was inserted into the V1 variable region of the 25S ribosomal gene. The RNA transcript from the transgene undergoes a series of maturation steps, including correct processing of the 5' end of 25S rRNA. Using primer extension analysis, expression of a complete rDNA repeat unit was compared with the activity of a repeat unit lacking a sequence called 'upstream Sal repeats'. No qualitative or quantitative differences were detected, suggesting that upstream repeat sequences of the rDNA intergenic region do not act as transcriptional enhancers for RNA polymerase L in A. thaliana.

Perturbation of the ubiquitin system causes leaf curling, vascular tissue alterations and necrotic lesions in a higher plant.

A ubiquitin variant with Lys48 changed to Arg acts in vitro as an inhibitor of ubiquitin dependent protein degradation. To assess the role of this proteolytic pathway in the life cycle of plants, we expressed the ubiquitin variant in Nicotiana tabacum. Expression of variant mono- or polyubiquitin leads to marked abnormalities in vascular tissue. In addition, overexpression of variant polyubiquitin induces discrete lesions on leaves. This indicates that perturbations of the ubiquitin system can induce a programmed necrotic response in plants.

Integration of common bean ( Phaseolus vulgaris L.) linkage and chromosomal maps.

Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.

The gag homologue of retrotransposon Ty1 assembles into spherical particles in Escherichia coli.

Expression of TyA (reading frame A) of the yeast retrotransposon Ty1 in Escherichia coli is possible by using efficient transcriptional and translational initiation signals. When expressed in E. coli, the gag homologue of Ty1 assembles into spherical particles similar, but not identical to virus-like particles in the natural host of Ty1, Saccharomyces cerevisiae. Deletion analysis reveals a domain in the C-terminus of TyA that is essential for the assembly process. These findings indicate that an early step of the retroelement life cycle, assembly of the gag homologue into spherical particles, does not depend on specific host factors. The experiments also demonstrate that Ty1 Gag fusion proteins, potential tools for immunization, can be produced in E. coli, an organism that lacks endogenous retrotransposons.

PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway.

Mutants in the PRT1 gene of Arabidopsis thaliana are impaired in the degradation of a normally short-lived intracellular protein that contains a destabilizing N-terminal residue. Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway. The chromosomal position of PRT1 was determined, and the PRT1 gene was isolated by map-based cloning. The 45-kDa PRT1 protein contains two RING finger domains and one ZZ domain. No other proteins in databases match these characteristics of PRT1. There is, however, a weak similarity to Rad18p of Saccharomyces cerevisiae. The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of PRT1 in the plant N-end rule pathway.

The Tpv2 family of retrotransposons of Phaseolus vulgaris: structure, integration characteristics, and use for genotype classification.

The Tpv2 family of transposable elements of common bean, Phaseolus vulgaris, belongs to the Ty1/copia group of long terminal repeat (LTR) containing retrotransposons. By reverse transcriptase (RT)-PCR and by analysis of genomic clones, we characterized four of the approximately 40 copies present in the Phaseolus genome, and the genomic environment of their integration sites. Tpv2 integrated preferentially into actively transcribed regions. While none of the isolated elements had all the functional domains necessary for transposition, analysis of bean cultivars suggested that some members of the Tpv2 family transposed in recent breeding history. Probes derived from Tpv2, as well as flanking genomic sequences, may be useful for classifying Phaseolus cultivars.

The 'prespore-like cells' of Dictyostelium have ceased to express a prespore gene: analysis using short-lived beta-galactosidases as reporters.

In transgenic strains of Dictyostelium discoideum that express beta-galactosidase under the control of a prespore-specific promoter, only early slugs show reporter confined to the prespore zone. As slugs migrate beta-galactosidase-positive cells accumulate in the prestalk zone; ultimately, there may be so many that the prestalk-prespore boundary is no longer distinguishable (Harwood, A., Early, A., Jermyn, K. and Williams, J. (1991) Differentiation 46, 7-13). It is not clear whether these 'anomalous' reporter-positive cells currently express prespore genes; another possibility is that they are ex-prespore cells that have transformed to prestalk and sorted to the prestalk zone (Sternfeld, J. (1993) Roux Archiv. Dev. Biol. 201, 354-363), while retaining their previously produced reporter. To test the activity of the prespore genes in these cells, we have made prespore reporter constructs whose products decay quickly; these are based on constructs used to investigate protein turnover in yeast (Bachmair, A., Finley, D. and Varshavsky, A. (1986) Science 234, 179-186). In strains bearing such constructs, beta-galactosidase-positive cells do not appear in the prestalk zone. The apparent deterioration of the prestalk/prespore pattern in older slugs is thus an artefact of reporter stability.

Universality and structure of the N-end rule.

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.