Cecile M. Pickart

Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme

The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage‐induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein‐‐its ability to mediate protein degradation dependent on the N‐end‐recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian E214k in E3 dependent ubiquitin‐‐protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl‐terminus of RAD6 is dispensable for the interaction with E3, and that purified S. cerevisiae E2(30k), product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.

E2/E3-mediated Assembly of Lysine 29-linked Polyubiquitin Chains

Polyubiquitin (Ub) chains linked through Lys-48–Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K m of ∼9 μm for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.

Ubiquitin Activation and Ligation

The energy dependence of intracellular protein turnover has been recognized for several decades. In the 1950s, Simpson1 and Schwieger et al. 2 showed that respiratory inhibitors decrease the rate of release of amino acids from cells. Therefore, these compounds, which decrease intracellular ATP concentration, inhibit intracellular protein turnover. Since peptide bond hydrolysis is a thermodynamically favorable process, why should protein break down require ATP? And how might ATP hydrolysis be coupled to peptide bond hydrolysis? Our understanding of the mechanistic basis for the ATP requirement has increased substantially in recent years. There are now several known ATP-dependent proteases. One of these is part of the multienzyme pathway that requires the small protein ubiquitin (Ub) as a cofactor. Although Ub-dependent protein breakdown is not the only ATP-dependent protein breakdown in higher eukaryotes,3 it is clear that the Ub-dependent pathway is quantitatively important. Turnover of at least 90% of the short-lived proteins in a mouse mammary carcinoma cell line is Ub dependent.4

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid‐ and solution‐phase immunochemical assays, employing anti‐ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13‐fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later‐stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse‐labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12‐fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin‐encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.

Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway. (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins. (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2s) except E2(20K) and E2(230K). Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted.

CHARACTERIZATION OF A CDNA CLONE ENCODING E2-20K, A MURINE UBIQUITIN-CONJUGATING ENZYME

The 20-kDa ubiquitin-conjugating enzyme E2-20K is induced specifically during a late stage of erythroid differentiation. Here we report the sequence of a murine cDNA encoding E2-20K. Northern blot analysis identified polyadenylated transcripts of 3.5 and 6.5 kb which are present at comparable levels in many nonerythroid tissues.

A reactive nucleophile proximal to vicinal thiols is an evolutionarily conserved feature in the mechanism of Arg aminoacyl-tRNA protein transferase☆

Aminoacyl-tRNA protein transferases post-translationally aminoacylate protein N-termini. At least in part, these enzymes function to allow a subset of cellular proteins to be targeted for protein degradation. A eukaryotic enzyme of this class, Arg aminoacyl-tRNA protein transferase, arginylates N-terminal Glu or Asp residues of proteins, allowing such proteins to be recognized by a specific ubiquitin-protein ligase. We showed previously that inorganic arsenite, a reagent expected to bind specifically to protein vicinal thiol groups, inhibited Arg aminoacyl-tRNA transferase activity in rabbit reticulocyte lysate (N. S. Klemperer and C. M. Pickart, 1989, J. Biol. Chem. 264, 19245-19252). We now report that a bifunctional arsenoxide reagent, p-[(bromoacetyl)-amino]phenylarsenoxide, is a potent and irreversible inactivator of the same enzyme (K0.5 = 11.5 microM). Bromoacetyl aniline, which lacks the arsenoxide moiety, has no effect. These results show that the transferase has a reactive nucleophile proximal to the site which binds arsenoxides. The related monofunctional arsenoxide reagent, p-aminophenylarsenoxide, is a reversible inhibitor whose potency (K0.5 = 7.7 microM) is 20-fold greater than that of inorganic arsenite. As expected for a mechanism in which p-aminophenylarsenoxide binds to vicinal thiol groups: (i) pretreatment of reticulocyte lysate with a thiol-blocking reagent prevents binding of the transferase to a phenylarsenoxide-Sepharose column; and (ii) inhibition by p-aminophenylarsenoxide is reversed by a competing chemical dithiol, but not by a monothiol reagent. Like the rabbit enzyme, Arg aminoacyl-tRNA protein transferase from the yeast Saccharomyces cerevisiae (expressed in Escherichia coli) is reversibly inhibited by the monofunctional phenylarsenoxide and irreversibly inactivated by the bifunctional phenylarsenoxide (but not by bromoacetylaniline). Thus, a reactive nucleophile proximal to vicinal thiol groups is a conserved feature of the activity of the transferase. We speculate that these groups are catalytic elements in the transferase active site.

Several mammalian ubiquitin carrier proteins, but not E220K, are related to the 20-kDA yeast E2, RAD6

Western blot analysis was used to probe the relationships between the multiple ubiquitin carrier proteins (E2 s) of rabbit reticulocytes and the 20-kDa E2 encoded by the RAD6 gene of the yeast S. cerevisiae. Reticulocyte E2-14K, E2-17K, and E2-25K each reacted with two or more polyclonal anti-RAD6 antibody preparations; E2-20K, E2-35K, and E2-230K did not cross-react. These results suggest that some, but not all, reticulocyte E2 s are members of a RAD6-like protein family which is conserved within and across species. RAD6 and E2-20K were also shown to multi-ubiquitinate histones by different mechanisms.