We report that Gcn4, a yeast transcriptional activator of the bZIP family involved in the regulation of the biosynthesis of amino acids and purines, is rapidly turned over. This degradation is inhibited under conditions of starvation for amino acids. Degradation is also inhibited by single amino acid alterations in a region adjacent to the Gcn4 activation domain. Furthermore, we show that degradation of Gcn4 proceeds through the ubiquitin pathway, a major proteolytic system for cytoplasmic proteins, and is dependent on two specific ubiquitin conjugating enzymes, Cdc34 (Ubc3) and Rad6 (Ubc2). As a first step towards reconstituting the Gcn4 degradation pathway in vitro, we show that purified Cdc34 and Rad6 proteins are able to direct the specific ubiquitination of Gcn4.

Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long‐lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step‐up is followed by a slower phase of proteolysis. During the first 2 h after temperature step‐up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step‐up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild‐type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat‐denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step‐up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

The p38 mitogen‐activated protein kinases are activated in response to various extracellular signals in eukaryotic cells and play a critical role in the cellular responses to these signals. The four mammalian isoforms (p38α, p38β, p38γ, and p38δ) are coexpressed and coactivated in the same cells. The exact role of each p38 isoform has not been entirely identified, in part due to the inability to activate each member individually. This could be resolved by the use of intrinsically active mutants. Based on previous studies on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001) J Biol Chem276, 25351–2538] and human p38α[Diskin R, Askari N, Capone R, Engelberg D & Livnah O (2004) J Biol Chem279, 47040–47049] we have generated intrinsically active p38β, p38γ and p38δ mutants. In addition, we have identified a new activating mutation site in p38α. Most of the activating mutations are located in the L16 loop, in which conformational changes were shown to induce activation. We show that these changes impose substantial autophosphorylation activity, providing a mechanistic explanation for the intrinsic activity of the mutants. The new active variants maintain specificity towards substrates and inhibitors similar to that of the parental wild‐type proteins, and are phosphorylated by mitogen‐activated protein kinase kinase 6, their upstream activator. Thus, we have completed the development of a series of intrinsically active mutants of all p38 isoforms. These active variants could now become powerful tools for the elucidating the activation mechanism and specific biological roles of each p38 isoform.

Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins. C hicken avidin and bacterial streptavidin are indispensable proteins for extensive types of biotechnological application. Avidin is found in egg whites of reptiles, amphibians, and birds, whereas streptavidin is found in the bacterium Streptomyces avidinii. The two proteins share many biochemical and structural features (1, 2), the foremost characteristic being that both bind biotin with extremely high affinity, representing the strongest noncovalent interaction known in nature (3).Although the two proteins share only 30% identity and 40% similarity in their sequence, their tertiary fold is highly similar: tetramers comprising four identical subunits, each of which contains a single biotin-binding site. The tertiary fold of avidin and streptavidin consists of an eight-stranded antiparallel ␤-barrel, and the main difference between the two protein lies in the size, composition, and conformation of the loops connecting the strands (4-6). The hairpin loop connecting strands ␤3 and ␤4 (the L3,4 loop) in both proteins plays an important role in biotin binding (7). In avidin, the loop is three residues larger than that of streptavidin. In the apoproteins, L3,4 in both avidin and streptavidin has an open or disordered conformation. Upon binding biotin, the loop closes in a lid-like manner, thus burying the ligand almost completely.The latter similarities are offset by relatively subtle differences in the two proteins. One st...