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Biomedical subjects

A Varshavsky

Publications and source records attributed to A Varshavsky.

At least 19 recordsLinked to original sources
PubMed2001

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Anaphase↗
PubMed2001

RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit.

The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t(1/2) approximately 2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Delta cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.

Binding Sites↗
PubMed2001

Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3alpha) of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

Adipose Tissue↗
PubMed2000

Detecting and measuring cotranslational protein degradation in vivo.

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.

Cysteine Endopeptidases↗
PubMed2000

RGS4 is arginylated and degraded by the N-end rule pathway in vitro.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.

Animals↗
PubMed2000

Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway.

Protein degradation by the ubiquitin system controls the intracellular concentrations of many regulatory proteins. A protein substrate of the ubiquitin system is conjugated to ubiquitin through the action of three enzymes, E1, E2 and E3, with the degradation signal (degron) of the substrate recognized by E3 (refs 1-3). The resulting multi-ubiquitylated substrate is degraded by the 26S proteasome. Here we describe the physiological regulation of a ubiquitin-dependent pathway through allosteric modulation of its E3 activity by small compounds. Ubr1, the E3 enzyme of the N-end rule pathway (a ubiquitin-dependent proteolytic system) in Saccharomyces cerevisiae mediates the degradation of Cup9, a transcriptional repressor of the peptide transporter Ptr2 (ref. 5). Ubr1 also targets proteins that have destabilizing amino-terminal residues. We show that the degradation of Cup9 is allosterically activated by dipeptides with destabilizing N-terminal residues. In the resulting positive feedback circuit, imported dipeptides bind to Ubr1 and accelerate the Ubr1-dependent degradation of Cup9, thereby de-repressing the expression of Ptr2 and increasing the cell's capacity to import peptides. These findings identify the physiological rationale for the targeting of Cup9 by Ubr1, and indicate that small compounds may regulate other ubiquitin-dependent pathways.

Allosteric Regulation↗
PubMed2000

Physical association of ubiquitin ligases and the 26S proteasome.

The ubiquitin (Ub) system recognizes degradation signals of the target proteins through the E3 components of E3-E2 Ub ligases. A targeted substrate bears a covalently linked multi-Ub chain and is degraded by the ATP-dependent 26S proteasome, which consists of the 20S core protease and two 19S particles. The latter mediate the binding and unfolding of a substrate protein before its transfer to the interior of the 20S core. It is unclear how a targeted substrate is delivered to the 26S proteasome, inasmuch as Rpn10p, the only known proteasomal subunit that binds multi-Ub chains, has been found to be not essential for degradation of many proteins in the yeast Saccharomyces cerevisiae. Here we show that Ubr1p and Ufd4p, the E3 components of two distinct Ub ligases, directly interact with the 26S proteasome. Specifically, Ubr1p is shown to bind to the Rpn2p, Rpt1p, and Rpt6p proteins of the 19S particle, and Ufd4p is shown to bind to Rpt6p. These and related results suggest that a substrate-bound Ub ligase participates in the delivery of substrates to the proteasome, because of affinity between the ligase's E3 component and specific proteins of the 19S particle.

Escherichia coli↗
PubMed2000

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

Amidohydrolases↗
PubMed1999

The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain.

We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system.

Amino Acid Sequence↗
PubMed1999

Degradation signals in the lysine-asparagine sequence space.

The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding.

Amino Acid Sequence↗
PubMed1999

Bivalent inhibitor of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.

Binding Sites↗
PubMed1999

The N-end rule pathway is required for import of histidine in yeast lacking the kinesin-like protein Cin8p.

The N-end rule pathway is a ubiquitin-dependent proteolytic system whose targets include proteins bearing destabilizing N-terminal residues. We carried out a synthetic lethal screen for Saccharomyces cerevisiae mutants that require the N-end rule pathway for cell viability. A mutant thus identified, termed sln2, could not grow in the absence of Ubr1p, the recognition component of the N-end rule pathway, which was not essential for viability of the parental strain under the same conditions. Further analysis showed that inviability of sln2ubr1Delta cells could be rescued either by the HIS3 gene (which was absent from the parental strain) or by a high concentration of histidine in the medium. This defect in histidine uptake, exhibited by the sln2 mutant in the absence but not in the presence of Ubr1p, was traced to the gene HIP1, which encodes the histidine transporter. HIP1 was underexpressed in sln2 ubr1Delta cells, in comparison to either sln2 UBR1 or SLN2 ubr1Delta cells. Yet another property of the sln2 mutant was its inviability at 37 degrees C, which could not be rescued by either UBR1 or HIS3. This feature of sln2 allowed the cloning of SLN2, which was found to be a gene called CIN8, encoding a kinesin-like protein. Thus, either the N-end rule pathway or Cin8p must be present for the viability-sustaining rate of histidine import in S. cerevisiae auxotrophic for histidine. We consider possible mechanisms of this previously unsuspected link between kinesins, ubiquitin-dependent proteolysis, and the import of histidine.

Base Sequence↗
PubMed1999

Analysis of a conditional degradation signal in yeast and mammalian cells.

The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues. The latter are a part of the degradation signal called the N-degron. Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts [a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety]. Previous work has shown that Arg-DHFRts is long-lived at 23 degreesC but short-lived at 37 degreesC in the yeast Saccharomyces cerevisiae. In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-Mr ligand of DHFR. In S. cerevisiae, Arg-DHFRts is degraded at 37 degreesC exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety. In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron. When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed eDeltaK, the resulting Arg-eDeltaK-DHFRts was rapidly degraded at both 23 degreesC and 37 degreesC. Moreover, the degradation of Arg-eDeltaK-DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degreesC results, at least in part, from steric inaccessibility of its N-terminal arginine. The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound. We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.

Animals↗
PubMed1999

Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum.

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

Base Sequence↗
PubMed1999

Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.

Alternative Splicing↗