Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, conside...

The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction

▪ Abstract In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. The 26S proteasome is a 2.5-MDa molecular machine built from ∼31 different subunits, which catalyzes protein degradation. It contains a barrel-shaped proteolytic core complex (the 20S proteasome), capped at one or both ends by 19S regulatory complexes, which recognize ubiquitinated proteins. The regulatory complexes are also implicated in unfolding and translocation of ubiquitinated targets into the interior of the 20S complex, where they are degraded to oligopeptides. Structure, assembly and enzymatic mechanism of the 20S complex have been elucidated, but the functional organization of the 19S complex is less well understood. Most subunits of the 19S complex have been identified, however, specific functions have been assigned to only a few. A low-resolution structure of the 26S proteasome has been obtained by electron microscopy, but the precise arrangement of subunits in the 19S co...

The 26S Proteasome: A Molecular Machine Designed for Controlled Proteolysis

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin-induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair-fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin-1/MAFbx and MuRF-1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down-regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up-regulated (lipin, metallothionein, AMP deaminase, RNA helicase-related protein, TG interacting factor) and several growth-related mRNAs were down-regulated (P311, JUN, IGF-1-BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fj.03-0610com

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression

The cellular stress response is a universal mechanism of extraordinary physiological/pathophysiological significance. It represents a defense reaction of cells to damage that environmental forces inflict on macromolecules. Many aspects of the cellular stress response are not stressor specific because cells monitor stress based on macromolecular damage without regard to the type of stress that causes such damage. Cellular mechanisms activated by DNA damage and protein damage are interconnected and share common elements. Other cellular responses directed at re-establishing homeostasis are stressor specific and often activated in parallel to the cellular stress response. All organisms have stress proteins, and universally conserved stress proteins can be regarded as the minimal stress proteome. Functional analysis of the minimal stress proteome yields information about key aspects of the cellular stress response, including physiological mechanisms of sensing membrane lipid, protein, and DNA damage; redox sensing and regulation; cell cycle control; macromolecular stabilization/repair; and control of energy metabolism. In addition, cells can quantify stress and activate a death program (apoptosis) when tolerance limits are exceeded.

Molecular and evolutionary basis of the cellular stress response

The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the environment were included. A total of 708 structural open reading frames (ORFs) were accounted for in the reconstructed network, corresponding to 1035 metabolic reactions. Further, 140 reactions were included on the basis of biochemical evidence resulting in a genome-scale reconstructed metabolic network containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to approximately 16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared with Escherichia coli. The reconstructed metabolic network is the first comprehensive network for a eukaryotic organism, and it may be used as the basis for in silico analysis of phenotypic functions.

/pdf/genome-scale-reconstruction-of-the-saccharomyces-cerevisiae-24mayjyh5r.pdf

Genome-Scale Reconstruction of the Saccharomyces cerevisiae Metabolic Network

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identitity with TKL1 [Sundstrom, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb18268.x

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2894%2901045-5

Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function

Characterization of the prephenate dehydrogenase-encoding gene, TYR1, from Saccharomyces cerevisiae.

In the framework of the EC programme for sequencing yeast chromosome II, we have determined the nucleotide sequence of a 70 kb region. Subsequent analysis revealed 35 open reading frames, 14 of which correspond to known yeast genes. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 1.98 kb. Coding regions occupy 75% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes. The complete nucleotide sequence of the 70 kb segment as depicted in Figure 1 has been deposited in the EMBL data library under Accession Number X78993.

Analysis of a 70 kb region on the right arm of yeast chromosome II.

We have analysed a region some 30 kb centromere distal form PHO5 on the right arm of yeast chromosome II and determined the nucleotide sequence of a 8.95 kb DNA segment from this region. By this analysis we were able to derive the precise location and the transcriptional orientation of CMD1, ALG1, SSN6 and LYS2. An open reading frame of 2370 bp was locatlized between SSN6 and LYS2, which has recently been identified (Schild et al., 1991) to be the RAD16 gene. The putative gene product, 790 amino acids in length, reveals several interesting freatures. It contains a nuclear target singnature and shares several blocks of similarity with the yeast recombinational repair protein RAD54 and the nuclear factor SNF2 (SW12), which is required for teh transcriptioal activation of a number of yeast genes. The similarity blocks in these three proteins are reminiscent of those found in the helicase superfamily. Furthrmore, RAD16 contains a novel ‘double-finger’ motif, which has been encountered in a variety of proteins from different organisms that are suggested to interact with DNA and are involved in diverse functions including site-specific recombination, DNA repair, and transcriptional regulation. The putative gene product of RAD16 then is the first example of a proteins in which the novel double-finger motif is found to be combined with a poteintial DNA helicase framework.

Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins.

Gertrud Mannhaupt

Papers

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function

Characterization of the prephenate dehydrogenase-encoding gene, TYR1, from Saccharomyces cerevisiae.

Analysis of a 70 kb region on the right arm of yeast chromosome II.

Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins.