Daniel Finley

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses

Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.

Ubiquitin Dependence of Selective Protein Degradation Demonstrated in the Mammalian Cell Cycle Mutant ts85

We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.

Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Ubiquitin, a 76 residue protein, occurs in eucaryotic cells either free or covalently joined to a variety of protein species. Previous work suggested that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. We show that the mouse cell line ts85 , a previously isolated cell cycle mutant, is temperature-sensitive in ubiquitin-protein conjugation, and that this effect is due to the specific thermolability of the ts85 ubiquitin-activating enzyme (E1). From E1 thermoinactivation kinetics in mixed (wild-type plus ts85 ) extracts, and from copurification of the determinant of E1 thermolability with E1 in ubiquitin-affinity chromatography, we conclude that the determinant of E1 thermolability is contained within the E1 polypeptide. ts85 cells fail to degrade otherwise short-lived intracellular proteins at the nonpermissive temperature (accompanying paper), demonstrating that degradation of the bulk of short-lived proteins in this higher eucaryotic cell proceeds through a ubiquitin-dependent pathway. We discuss possible roles of ubiquitin-dependent pathways in DNA transactions, the cell cycle, and the heat shock response.

The ubiquitin system: functions and mechanisms

Abstract Ubiquitin is a protein found in eukaryotic cells either free or covalently joined to a variety of cytoplasmic and nuclear proteins. Recent biochemical and genetic evidence indicated that conjugation of ubiquitin to short-lived proteins is essential for their selective degradation ( in vivo ). Also discussed here are recent mechanistic studies of ubiquitin-dependent proteolysis, the unusual organization of the ubiquitin gene, and putative roles of the ubiquitin system in chromosome function and the stress response.

Enhancement of immunoblot sensitivity by heating of hydrated filters

Immunoblots of either dot or Western type were exposed to heat before reaction with antibody. Dramatic increases in immunoblot sensitivity were seen for certain antigen-antibody pairs after heating of either dry or hydrated nitrocellulose filters at or above 100 degrees C. Heating of filters in the hydrated state improved the linearity of immunodetection and produced the highest signal-to-noise ratio. This treatment greatly increased immunoblot sensitivity with several peptide-generated antibodies, whereas decreased sensitivity was seen with antibodies against native proteins. Heating of hydrated filters after antigen immobilization is thus a potentially powerful way to increase the sensitivity of immunoblot analysis for antibodies that preferentially recognize epitopes in denatured proteins.

Molecular Genetics of the Ubiquitin System

The ubiquitin (Ub) system was first approached experimentally using purely biochemical methods.1 Genetic techniques provide not only an alternative way to address previously posed questions in this field2,3 but also a strategy for the identification and dissection of the physiological functions of Ub. The genetic approach has turned up new components of the Ub system, such as polyubiquitin4–12 and the Ub-hybrid proteins,12–17 and more recently has led to identification of the product of the yeast DNA repair gene RAD6 as a Ub-conjugating enzyme.18 Our current studies are focused primarily on yeast, whose Ub system, as described in Section 3, closely resembles that of mammals. Thus, insights gained from molecular genetic analysis of the yeast system should be relevant to other eukaryotes as well. While Ub genes have now been cloned from a number of organisms (reviewed in refs. 19 and 20), mutational analysis of the Ub system has been restricted to yeast21 and the mammalian cell line ts85.2,3,22

The N-End Rule of Selective Protein Turnover

In both bacterial and eukaryotic cells, relatively long-lived proteins, whose half-lives are close to or exceed the cell generation time, coexist with proteins whose half-lives can be less than 1% of the cell generation time. Rates of intracellular protein degradation are a function of the cell’s physiological state and appear to be controlled differentially for individual proteins.1–7 In particular, damaged and some otherwise abnormal proteins are metabolically unstable.1–10 It is also clear that many otherwise undamaged regulatory proteins are extremely short-lived in vivo.1–23 Metabolic instability of such proteins allows for efficient temporal control of their intracellular concentrations through regulated changes in rates of their synthesis or degradation. Instances in which the metabolic instability of an intracellular protein is known to be directly relevant to its function include the cII protein of bacteriophage λ (cII is the essential component of a molecular switch that determines whether λ grows lytically or lysogenizes an infected cell),16–18 the σ32 factor of the Escherichia coli RNA polymerase (σ32 confers on RNA polymerase the specificity for promoters of heat-shock genes),15,19–21 and the HO endonuclease of the yeast Saccharomyces cerevisiae (HO is a site-specific endodeoxyribonuclease that initiates the process of mating-type interconversion in yeast).22,23