Evolution of Mutational Robustness in the Yeast Genome: A Link to Essential Genes and Meiotic Recombination Hotspots

Deleterious mutations inevitably emerge in any evolutionary process and are speculated to decisively influence the structure of the genome. Meiosis, which is thought to play a major role in handling mutations on the population level, recombines chromosomes via non-randomly distributed hot spots for meiotic recombination. In many genomes, various types of genetic elements are distributed in patterns that are currently not well understood. In particular, important (essential) genes are arranged in clusters, which often cannot be explained by a functional relationship of the involved genes. Here we show by computer simulation that essential gene (EG) clustering provides a fitness benefit in handling deleterious mutations in sexual populations with variable levels of inbreeding and outbreeding. We find that recessive lethal mutations enforce a selective pressure towards clustered genome architectures. Our simulations correctly predict (i) the evolution of non-random distributions of meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers and EG clustering, (iii) the evolution of EG enrichment in pericentromeric regions and (iv) the associated absence of meiotic crossovers (cold centromeres). Our results furthermore predict optimal crossover rates for yeast chromosomes, which match the experimentally determined rates. Using a Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal phenotypes result predominantly from mutation of single loci and generally do not impair mating, which leads to an accumulation of mutational load following meiosis and mating. We hypothesize that purging of deleterious mutations in essential genes constitutes an important factor driving meiotic crossover. Therefore, the increased robustness of populations to deleterious mutations, which arises from clustered genome architectures, may provide a significant selective force shaping crossover distribution. Our analysis reveals a new aspect of the evolution of genome architectures that complements insights about molecular constraints, such as the interference of pericentromeric crossovers with chromosome segregation

Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving

Synthetic chemists have devoted tremendous effort towards the production of precision synthetic polymers with defined sequences and specific functions. However, the creation of a general technology that enables precise control over monomer sequence, with efficient isolation of the target polymers, is highly challenging. Here, we report a robust strategy for the production of sequence-defined synthetic polymers through a combination of liquid-phase synthesis and selective molecular sieving. The polymer is assembled in solution with real-time monitoring to ensure couplings proceed to completion, on a three-armed star-shaped macromolecule to maximize efficiency during the molecular sieving process. This approach is applied to the construction of sequence-defined polyethers, with side-arms at precisely defined locations that can undergo site-selective modification after polymerization. Using this versatile strategy, we have introduced structural and functional diversity into sequence-defined polyethers, unlocking their potential for real-life applications in nanotechnology, healthcare and information storage

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation

Sec61p is part of the endoplasmic reticulum-associated degradation machinery

Endoplasmic reticulum-associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro-translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd-Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins

Degradation of unassembled Vph1p reveals novel aspects of the yeast ER quality control system

The endoplasmic reticulum quality control (ERQC) system retains and degrades soluble and membrane proteins that misfold or fail to assemble. Vph1p is the 100 kDa membrane subunit of the yeast Saccharomyces cerevisiae V–ATPase, which together with other subunits, assembles into the V–ATPase in the ER, requiring the ER resident protein Vma22p. In vma22Δ cells, Vph1p remains an integral membrane protein with wild-type topology in the ER membrane before undergoing a rapid and concerted degradation requiring neither vacuolar proteases nor transport to the Golgi. Failure to assemble targets Vph1p for degradation in a process involving ubiquitylation, the proteasome and cytosolic but not ER lumenal chaperones. Vph1p appears to possess the traits of a ‘classical’ ERQC substrate, yet novel characteristics are involved in its degradation: (i) UBC genes other than UBC6 and UBC7 are involved and (ii) components of the ERQC system identified to date (Der1p, Hrd1p/Der3p and Hrd3p) are not required. These data suggest that other ERQC components must exist to effect the degradation of Vph1p, perhaps comprising an alternative pathway

The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment

Misfolded proteins of the endoplasmic reticulum (ER) are targeted to the cytoplasm for proteasomal degradation. Key components of this process are ER membrane-bound ubiquitin ligases. These ligases associate with the cytoplasmic AAA-ATPase Cdc48p/p97, which is thought to support the release of malfolded proteins from the ER. Here, we characterize a yeast protein complex containing the ubiquitin ligase Hrd1p and the ER membrane proteins Hrd3p and Der1p. Hrd3p binds malfolded proteins in the ER lumen enabling their delivery to downstream components. Therefore, we propose that Hrd3p acts as a substrate recruitment factor for the Hrd1p ligase complex. Hrd3p function is also required for the association of Cdc48p with Hrd1p. Moreover, our data demonstrate that recruitment of Cdc48p depends on substrate processing by the Hrd1p ligase complex. Thus, the Hrd1p ligase complex unites substrate selection in the ER lumen and polyubiquitination in the cytoplasm and links these processes to the release of ER proteins via the Cdc48p complex