Nonpermissive Research Articles

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

BackgroundPest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required.ResultsHere we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster.ConclusionsWe have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient.

Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

Cold tolerance, water balance, energetics, gas exchange, and diapause in overwintering brown marmorated stink bugs

Halyomorpha halys (Hemiptera: Pentatomidae) is an emerging pest which established in Ontario, Canada, in 2012. Halyomporpha halys overwinters in anthropogenic structures as an adult. We investigated seasonal variation in the cold tolerance, water balance, and energetics of H. halys in southwestern Ontario. We also induced diapause in laboratory-reared animals with short daylength at permissive temperatures and compared cold tolerance, water balance, energetics, and metabolism and gas exchange between diapausing and non-diapausing individuals. Halyomorpha halys that overwintered outside in Ontario all died, but most of those that overwintered in sheltered habitats survived. We confirm that overwintering H. halys are chill-susceptible. Over winter, Ontario H. halys depressed their supercooling point to c. −15.4 °C, and 50% survived a 1 h exposure to −17.5 °C. They reduce water loss rates over winter, and do not appear to significantly consume lipid or carbohydrate reserves to a level that might cause starvation. Overall, it appears that H. halys is dependent on built structures and other buffered microhabitats to successfully overwinter in Ontario. Laboratory-reared diapausing H. halys have lower supercooling points than their non-diapausing counterparts, but LT50 is not enhanced by diapause induction. Diapausing H. halys survive desiccating conditions for 3–4 times longer than those not in diapause, through decreases in both respiratory and cuticular water loss. Diapausing H. halys do not appear to accumulate any more lipid or carbohydrate than those not in diapause, but do have lower metabolic rates, and are slightly more likely to exhibit discontinuous gas exchange.

Inhibition of PAR-1 delays aging via activating AMPK in C. elegans.

The antagonistic pleiotropy theory of aging suggests that genes essential for growth and development are likely to modulate aging later in life. Previous studies in C. elegans demonstrate that inhibition of certain developmentally essential genes during adulthood leads to significant lifespan extension. PAR-1, a highly conserved serine/threonine kinase, functions as a key cellular polarity regulator during the embryonic development. However, the role of PAR-1 during adulthood remains unknown. Here we show that inhibition of par-1 either by a temperature-sensitive mutant or by RNAi knockdown only during adulthood is sufficient to extend lifespan in C. elegans. Inhibition of par-1 also improves healthspan, as indicated by increased stress resistance, enhanced proteotoxicity resistance, as well as reduced muscular function decline over time. Additionally, tissue-enriched RNAi knockdown analysis reveals that PAR-1 mainly functions in the epidermis to regulate lifespan. Further genetic epistatic and molecular studies demonstrate that the effect of par-1 on lifespan requires the AMP-activated protein kinase (AMPK), and RNAi knockdown of par-1 results in age-dependent AMPK activation and reduced lipid accumulation in the metabolic tissue. Taken together, our findings reveal a previously undescribed function of PAR-1 in adulthood, which will help to understand the molecular links between development and aging.

The Role of Water Temperature Modelling in the Development of a Release Strategy for Cyprinid Herpesvirus 3 (CyHV-3) for Common Carp Control in Southeastern Australia

The common carp (Cyprinus carpio) is an invasive species in the rivers and waterways of southeastern Australia, and it has been implicated in the serious decline of many native fish species. Over the past 50 years, various control options have been explored, and to date, these have been ineffective or cost-prohibitive. Most recently, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a biocontrol agent because of its high specificity and mortality rate. However, the virus is known to be only effective in a permissive water temperature range of approximately 16–28 °C. To define when this occurs, we undertook a hydrological reconstruction of five diverse river catchments (>130,000 km2) of southeastern Australia over three decades. This confirmed, in the studied areas, that while water temperatures are permissive from spring through to autumn, the time of year that this starts and ends is highly variable, interannually, and with strong latitudinal and altitudinal gradients between and within catchments. The results show that the virus should be effective with respect to water temperature throughout the water temperature range that carp occur in most of southeastern Australia. However, detailed water temperature estimation would still be required to determine the exact week of the start of release in any given catchment. Referring to observations in wild carp populations, we point out the limitation of developing a “release strategy” based solely on water temperature modelling and the need to incorporate fish biology and ecology into this planning.

Seroconversion and Skin Mucosal Parameters during Koi Herpesvirus Shedding in Common Carp, Cyprinus carpio.

Seroconversion and the mucosal lysozyme G (lysG), complement 3 (c3), and immunoglobulins M (IgMsec) and Z2 (IgZ2) were measured for up to 900 degree days (DD) in skin swabs from common carp exposed to koi herpesvirus (KHV or CyHV-3) at either a non-permissive temperature (12 °C) or permissive temperatures (17 and 22 °C), and in survivors subjected to temperature increase to 22 °C 500 DD after the initial exposure. The survival rate at 22 °C varied from 100% in fish initially exposed at 12 °C, to 20% at 17 °C and 0% at 22 °C. Viral shedding episodes lasted for up to 29 days (493 DD) for fish clinically infected at 17 °C, and up to 57 days (684 DD) for asymptomatic fish held at 12 °C. Up-regulation of lysG transcripts was measured at 17 and 22 °C. Down-regulation of c3 and IgMsec transcripts was measured independent of the water temperature, followed by up-regulation after the temperature increase coinciding with seroconversion and clearance of KHV from the skin mucus. IgZ2 mRNA showed a negative correlation with IgM transcripts. KHV subversion of the complement system at the mucosal site coupled with poor immunoglobulin secretion during the viral replication might contribute to the long window of viral shedding, thus facilitating viral transmission.

A temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of α-maltose-1-phosphate

BackgroundThe bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. MethodsWe reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. ResultsThe mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. ConclusionsWe have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. General significanceThis study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.

SiCTeC: An inexpensive, easily assembled Peltier device for rapid temperature shifting during single-cell imaging.

Single-cell imaging, combined with recent advances in image analysis and microfluidic technologies, have enabled fundamental discoveries of cellular responses to chemical perturbations that are often obscured by traditional liquid-culture experiments. Temperature is an environmental variable well known to impact growth and to elicit specific stress responses at extreme values; it is often used as a genetic tool to interrogate essential genes. However, the dynamic effects of temperature shifts have remained mostly unstudied at the single-cell level, due largely to engineering challenges related to sample stability, heatsink considerations, and temperature measurement and feedback. Additionally, the few commercially available temperature-control platforms are costly. Here, we report an inexpensive (<$110) and modular Single-Cell Temperature Controller (SiCTeC) device for microbial imaging—based on straightforward modifications of the typical slide-sample-coverslip approach to microbial imaging—that controls temperature using a ring-shaped Peltier module and microcontroller feedback. Through stable and precise (±0.15°C) temperature control, SiCTeC achieves reproducible and fast (1–2 min) temperature transitions with programmable waveforms between room temperature and 45°C with an air objective. At the device’s maximum temperature of 89°C, SiCTeC revealed that Escherichia coli cells progressively shrink and lose cellular contents. During oscillations between 30°C and 37°C, cells rapidly adapted their response to temperature upshifts. Furthermore, SiCTeC enabled the discovery of rapid morphological changes and enhanced sensitivity to substrate stiffness during upshifts to nonpermissive temperatures in temperature-sensitive mutants of cell-wall synthesis enzymes. Overall, the simplicity and affordability of SiCTeC empowers future studies of the temperature dependence of single-cell physiology.

Genetic profiling of protein burden and nuclear export overload.

Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.

Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in an application that we refer to as mutant thermal proteome profiling (mTPP). This study characterized global impacts of temperature sensitivity-inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is a precise approach to measure changes in missense mutant-containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.

Evidence of positive regulation of mycobacteriophage D29 early gene expression obtained from an investigation using a temperature-sensitive mutant of the phage.

Mycobacteriophages are phages that infect and kill Mycobacteria, several of which, Mycobacterium tuberculosis (Mtb), for example, cause the disease tuberculosis. Although genomes of many such phages have been sequenced, we have very little insight into how they express their genes in a controlled manner. To address this issue, we have raised a temperature-sensitive (ts) mutant of phage D29 that can grow at 37°C but not at 42°C and used it to perform differential gene expression and proteome analysis studies. Our analysis results indicate that expression of genes located in the right arm, considered to be early expressed, was lowered as the temperature was shifted from 37°C to 42°C. In contrast, expression of those on the left, the late genes were only marginally affected. Thus, we conclude that transcription of genes from the two arms takes place independently of each other and that a specific factor must be controlling the expression of the right arm genes. We also observe that within the right arm itself; there exists a mechanism to ensure high-level synthesis of Gp48, a thymidylate synthase X. Enhanced presence of this protein in infected cells results in delayed lysis and higher phage yields.

Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2

The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160.

Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast Rpc160 to single Pol III leukodystrophy mutations in the pore domain can be overcome by a second mutation in the domain.

Inhibition of the alternative lengthening of telomeres pathway by subtelomeric sequences in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, telomerase is constitutively active and is essential for chromosome end protection and illimited proliferation of cell populations. However, upon inactivation of telomerase, alternative mechanims of telomere maintenance allow proliferation of only extremely rare survivors. S. cerevisiae type I and type II survivors differ by the nature of the donor sequences used for repair by homologous recombination of the uncapped terminal TG1-3 telomeric sequences. Type I amplifies the subtelomeric Y' sequences and is more efficient than type II, which amplifies the terminal TG1-3 repeats. However, type II survivors grow faster than type I survivors and can easily outgrow them in liquid cultures. The mechanistic interest of studying S. cerevisiae telomeric recombination is reinforced by the fact that type II recombination is the equivalent of the alternative lengthening of telomeres (ALT) pathway that is used by 5–15 % of cancer types as an alternative to telomerase reactivation. In budding yeast, only around half of the 32 telomeres harbor Y' subtelomeric elements. We report here that in strains harboring Y' elements on all telomeres, type II survivors are not observed, most likely due to an increase in the efficiency of type I recombination. However, in a temperature-sensitive cdc13-1 mutant grown at semi-permissive temperature, the increased amount of telomeric TG1-3 repeats could overcome type II inhibition by the subtelomeric Y’ sequences. Strikingly, in the 100 % Y’ strain the replicative senescence crisis normally provoked by inactivation of telomerase completely disappeared and the severity of the crisis was proportional to the percentage of chromosome-ends lacking Y’ subtelomeric sequences. The present study highlights the fact that the nature of subtelomeric elements can influence the selection of the pathway of telomere maintenance by recombination, as well as the response of the cell to telomeric damage caused by telomerase inactivation.

Protein stable isotope probing with H218O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12

Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins. We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode. We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response. Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities.

Computational studies of the properties and activities of selected trisubstituted benzimidazoles as potential antitubercular drugs inhibiting MTB-FtsZ polymerization

Trisubstituted benzimidazoles (trisbenz) are significantly active against nonreplicating Mycobacterium tuberculosis (MTB) by inhibiting the polymerization of Filamentous Temperature Sensitive Mutant Z (FtsZ), an essential bacteria cell division protein. In-depth in-silico study of 5 of the most active trisubstituted benzimidazoles; trisbenz 1, 2, 3, 4 and 5, giving insight into their properties, such as stability, bioavailability, interactions with residues at the binding site of MTB-FtsZ and their influence on structural dynamics of the protein have been conducted. This was achieved through the application of in-silico methods including density functional theory (DFT) calculations, ADME properties calculations, molecular docking and molecular dynamics simulations. A DFT approach was applied to predict reactivity properties of potent FtsZ inhibitors, and the results reveal the relative reactivity of these inhibitors as bioactive moieties. The estimated ADME properties predicted all 5 compounds to be bioavailable and suitable for oral administration. Molecular docking, binding free energy, RMSD, RMSF, and hydrogen bond analysis confirmed these 5 compounds as potent MTB-FtsZ inhibitors. Although analyses proved these compounds to be bioactive and potent MTB-FtsZ inhibitors, however, trisbenz 1 appeared to be the most active against this protein while trisbenz 5 was the least active. This study further confirms the experimental study while also giving insight on the compounds mechanism of action and presents their bioavailability properties.

A Missense Mutation in a Large Subunit of Ribonucleotide Reductase Confers Temperature-Gated Tassel Formation.

Temperature is a major factor regulating plant growth. To reproduce at extreme temperatures, plants must develop normal reproductive organs when exposed to temperature changes. However, little is known about the underlying molecular mechanisms. Here, we identified the maize (Zea mays) mutant thermosensitive vanishing tassel1-R (tvt1-R), which lacks tassels at high (restrictive) temperatures due to shoot apical meristem (SAM) arrest, but forms normal tassels at moderate (permissive) temperatures. The critical stage for phenotypic conversion in tvt1-R mutants is V2 to V6 (Vn, where "n" is the number of leaves with collars visible). Positional cloning and allelism and complementation tests revealed that a G-to-A mutation causing a Arg277-to-His277 substitution in ZmRNRL1, a ribonucleotide reductase (RNR) large subunit (RNRL), confers the tvt1-R mutant phenotype. RNR regulates the rate of deoxyribonucleoside triphosphate (dNTP) production for DNA replication and damage repair. By expression, yeast two-hybrid, RNA sequencing, and flow cytometric analyses, we found that ZmRNRL1-tvt1-R failed to interact with all three RNR small subunits at 34°C due to the Arg277-to-His277 substitution, which could impede RNR holoenzyme (α2β2) formation, thereby decreasing the dNTP supply for DNA replication. Decreased dNTP supply may be especially severe for the SAM that requires a continuous, sufficient dNTP supply for rapid division, as demonstrated by the SAM arrest and tassel absence in tvt1-R mutants at restrictive temperatures. Our study reveals a novel mechanism of temperature-gated tassel formation in maize and provides insight into the role of RNRL in SAM maintenance.

The intermembrane space protein Mix23 is a novel stress-induced mitochondrial import factor

The biogenesis of mitochondria requires the import of hundreds of precursor proteins. These proteins are transported post-translationally with the help of chaperones, meaning that the overproduction of mitochondrial proteins or the limited availability of chaperones can lead to the accumulation of cytosolic precursor proteins. This imposes a severe challenge to cytosolic proteostasis and triggers a specific transcription program called the mitoprotein-induced stress response, which activates the proteasome system. This coincides with the repression of mitochondrial proteins, including many proteins of the intermembrane space. In contrast, herein we report that the so-far-uncharacterized intermembrane space protein Mix23 is considerably up-regulated when mitochondrial import is perturbed. Mix23 is evolutionarily conserved and a homolog of the human protein CCDC58. We found that, like the subunits of the proteasome, Mix23 is under control of the transcription factor Rpn4. It is imported into mitochondria by the mitochondrial disulfide relay. Mix23 is critical for the efficient import of proteins into the mitochondrial matrix, particularly if the function of the translocase of the inner membrane 23 is compromised such as in temperature-sensitive mutants of Tim17. Our observations identify Mix23 as a novel regulator or stabilizer of the mitochondrial protein import machinery that is specifically up-regulated upon mitoprotein-induced stress conditions.

Temperature Sensitive Photosynthesis: Point Mutated CEF-G, PRK, or PsbO Act as Temperature-Controlled Switches for Essential Photosynthetic Processes

Temperature sensitive mutants have been widely used to study structure, biogenesis and function of a large variety of essential proteins. However, this method has not yet been exploited for the study of photosynthesis. We used negative selection to isolate temperature-sensitive-photoautotrophic (TSP) mutants in Chlamydomonas reinhardtii. From a population of randomly mutagenized cells (n=12,000), a significant number of TSP mutants (n=157) were isolated. They were able to grow photoautotrophically at 25°C, but lacked this ability at 37°C. Further phenotypic characterization of these mutants enabled the identification of three unique and highly interesting mutant strains. Following, the selected strains were genetically characterized by extensive crossing and whole genome sequencing. Correspondingly, the single amino acid changes P628F in the Chloroplast-Elongation-Factor-G (CEF-G), P129L in Phosphoribulokinase (PRK), and P101H in an essential subunit of Photosystem II (PsbO) were identified. These key changes alter the proteins in such way that they were functional at the permissive temperature, however, defective at the restrictive temperature. These mutants are presented here as superb and novel tools for the study of a wide range of aspects relevant to photosynthesis research, tackling three distinct and crucial photosynthetic processes: Chloroplast translation, PET-chain, and CBB-cycle.