Proteasome regulation of petite-negativity in fission yeast.

Mitochondria carry out essential functions in eukaryotic cells. The mitochondrial genome encodes factors critical to support oxidative phosphorylation and mitochondrial protein import necessary for these functions. However, organisms like budding yeast can readily lose their mitochondrial genome, yielding respiration-deficient petite mutants. The fission yeast Schizosaccharomyces pombe is petite-negative, but some nuclear mutations enable the loss of its mitochondrial genome. Here, we characterize the classical petite -positive mutation ptp1-1 as a loss of function allele of the proteasome 19S regulatory subunit component mts4/rpn1 , involved in the Ubiquitin-dependent degradation pathway. The mutation results in an altered oxidative stress response, with increased levels of oxidized glutathione, and increased levels of mitochondrial and cytoplasmic chaperones. We propose that Ubiquitin-proteasome regulation of chaperones involved in the Unfolded Protein Response and mitochondrial protein import underlies petite-negativity in fission yeast.

Transposon Removal Reveals Their Adaptive Fitness Contribution.

Transposable elements are molecular parasites that persist in their host genome by generating new copies to outpace natural selection. Transposable elements exert a large influence on host genome evolution, in some cases providing adaptive changes. Here we measure the fitness effect of the transposable element insertions in the fission yeast Schizosaccharomyces pombe type strain by removing all insertions of its only native transposable element family, the long terminal repeat retrotransposon Tf2. We show that Tf2 elements provide a positive fitness contribution to its host. Tf2 ablation results in changes to the regulation of a mitochondrial gene and, consistently, the fitness effect are sensitive to growth conditions. We propose that Tf2 influences host fitness in a directed manner by dynamically rewiring the transcriptional response to metabolic stress.

Arrested replication forks guide retrotransposon integration.

Long terminal repeat (LTR) retrotransposons are an abundant class of genomic parasites that replicate by insertion of new copies into the host genome. Fungal LTR retrotransposons prevent mutagenic insertions through diverse targeting mechanisms that avoid coding sequences, but conserved principles guiding their target site selection have not been established. Here, we show that insertion of the fission yeast LTR retrotransposon Tf1 is guided by the DNA binding protein Sap1 and that the efficiency and location of the targeting depend on the activity of Sap1 as a replication fork barrier. We propose that Sap1 and the fork arrest it causes guide insertion of Tf1 by tethering the integration complex to target sites.

Evolutionarily conserved sites in yeast tropomyosin function in cell polarity, transport and contractile ring formation.

Tropomyosin is a coiled-coil protein that binds and regulates actin filaments. The tropomyosin gene in Schizosaccharomyces pombe, cdc8, is required for formation of actin cables, contractile rings, and polar localization of actin patches. The roles of conserved residues were investigated in gene replacement mutants. The work validates an evolution-based approach to identify tropomyosin functions in living cells and sites of potential interactions with other proteins. A cdc8 mutant with near-normal actin affinity affects patch polarization and vacuole fusion, possibly by affecting Myo52p, a class V myosin, function. The presence of labile residual cell attachments suggests a delay in completion of cell division and redistribution of cell patches following cytokinesis. Another mutant with a mild phenotype is synthetic negative with GFP-fimbrin, inferring involvement of the mutated tropomyosin sites in interaction between the two proteins. Proteins that assemble in the contractile ring region before actin do so in a mutant cdc8 strain that cannot assemble condensed actin rings, yet some cells can divide. Of general significance, LifeAct-GFP negatively affects the actin cytoskeleton, indicating caution in its use as a biomarker for actin filaments.

A molecular evolution approach to study the roles of tropomyosin in fission yeast.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

The flexible attachment of the N-domains to the ClpA ring body allows their use on demand.

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a Delta N variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9.

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.