The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry-based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria

Structural basis for the interaction of the chaperone Cbp3 with newly synthesized cytochrome b during mitochondrial respiratory chain assembly

Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3-Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol-cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein

The mitoribosome as a hub for integrating mitochondrial gene expression : Regulation of translation and early assembly of cytochrome b

The mitochondrial proteome is a mosaic of proteins from two different genetic systems. The majority of mitochondrial proteins are encoded in the nuclear genome and are synthesized in the cytosol before imported to mitochondria. Interestingly, a handful of mitochondrial proteins are still encoded in the small mitochondrial genome, which is a remnant of its bacterial origin. These proteins are almost exclusively highly hydrophobic core subunits of the respiratory chain and ATP synthase. To synthesize these proteins, mitochondria contain its own complete gene expression system including factors for replication, transcription and translation. During the course of evolution, these machineries have diverged from its origin and have acquired organelle-specific characteristics. Mitochondrially encoded proteins are synthesized on the specialized and membrane-tethered mitoribosome. To avoid stoichiometric problems during assembly, translation in mitochondria needs to be coordinated with import of nuclear encoded subunits. However, the mechanisms behind regulation of mitochondrial protein biogenesis are still largely unclear. My research has focused on mitochondria in the yeast Saccharomyces cerevisiae. This provides a possibility to manipulate genes in both the nuclear and mitochondrial genomes to study effects on mitochondrial translation and respiration. In this thesis, I present results that contributes to our understanding of how translation regulation is connected to early assembly of mitochondrially encoded proteins. Using a proximity labelling technique called Bio-ID, we constructed a proximity network of proteins covering the whole mitochondrial gene expression system. This revealed a close association of factors involved in transcription, translation, membrane insertion and assembly, with the mitoribosome as a central hub. In particular, the mitoribosomal polypeptide tunnel exit was found to host factors involved in both regulation of translation and early assembly of nascent polypeptides. Further analysis of the tunnel exit proximity interactome resulted in identification of the membrane protein Mrx4 as a novel repressor of cytochrome b (Cytb) synthesis. Cytb is a catalytic subunit of the respiratory complex III and is encoded by the gene COB on the mtDNA. Translation of COB is under control of the regulatory proteins Cbp1, Cbs1, Cbs2, and Cbp3-Cbp6. Mrx4 is shown to interact with Cbs2, likely to sequester COB mRNA at the tunnel exit in a repressed state, until binding of Cbp3-Cbp6 releases Cbs2 and triggers activation of COB translation. Cbp3-Cbp6 then interacts with the newly synthesized Cytb, as it emerges from the tunnel exit, to stabilize the nascent polypeptide in an early assembly intermediate. The thesis further describes this chaperone function of Cbp3-Cbp6 on Cytb using structural models and site-specific photo-crosslinking. The results indicated that conformational changes in Cytb, upon incorporation of heme bH, releases Cbp3-Cbp6 to activate a new round of COB translation. In summary, the work presented in this thesis demonstrates a high level of organization and novel connectivity of mitochondrial gene expression. In addition, it expands on our knowledge regarding the intricate feedback loop regulation of Cytb biogenesis and the importance to coordinate mitochondrial translation with import of nuclear encoded subunits.

Fastighetsmäklarbranschen - en relationsskapande bransch?

Validerat; 20101217 (root

Building brand and market communications in Swedish Football Clubs : A case study on BK Forward & KIF Örebro

Background                      European football has during the past decades seen a fast development towards a more professionalized and commercialized climate. This had led to the fact that a lot of clubs are run like any other company, and the focus on marketing and market shares has increased. To be able to create a strong brand clubs have to put a lot of work into marketing, not only of their own brand, but of their sponsors and partners as well. Purpose                              The purpose of this study is to (in step 1) analyze how two smaller Swedish football clubs build their brand and work with their marketing communication. In step 2, the purpose is to create a model of how these clubs may work to develop their brand and marketing communication. Method                              With a qualitative approach, the authors of this paper have analyzed text and documents as well as performed two in-depth interviews. Findings                             Both clubs lack a well-developed strategy regarding their work with their brand and marketing communication. However, there are in both cases factors that, if developed correctly, could help build a stronger brand and better the marketing communication. One of these factors is the clubs’ work with social responsibility. Conclusion                         There is an importance of creating a strong internal identity in order to be able to position the club and define the offer towards different segments. Through this comes the possibility to create a strong strategy for marketing communication

Rapid measurement of heteronuclear transverse relaxation rates using non-uniformly sampled R1ρ accordion experiments

Multidimensional, heteronuclear NMR relaxation methods are used extensively to characterize the dynamics of biological macromolecules. Acquisition of relaxation datasets on proteins typically requires significant measurement time, often several days. Accordion spectroscopy offers a powerful means to shorten relaxation rate measurements by encoding the “relaxation dimension” into the indirect evolution period in multidimensional experiments. Time savings can also be achieved by non-uniform sampling (NUS) of multidimensional NMR data, which is used increasingly to improve spectral resolution or increase sensitivity per unit time. However, NUS is not commonly implemented in relaxation experiments, because most reconstruction algorithms are inherently nonlinear, leading to problems when estimating signal intensities, relaxation rate constants and their error bounds. We have previously shown how to avoid these shortcomings by combining accordion spectroscopy with NUS, followed by data reconstruction using sparse exponential mode analysis, thereby achieving a dramatic decrease in the total length of longitudinal relaxation experiments. Here, we present the corresponding transverse relaxation experiment, taking into account the special considerations required for its successful implementation in the framework of the accordion-NUS approach. We attain the highest possible precision in the relaxation rate constants by optimizing the NUS scheme with respect to the Cramér-Rao lower bound of the variance of the estimated parameter, given the total number of sampling points and the spectrum-specific signal characteristics. The resulting accordion-NUS R1ρ relaxation experiment achieves comparable precision in the parameter estimates compared to conventional CPMG (Carr-Purcell-Meiboom-Gill) R2 or spin-lock R1ρ experiments while saving an order of magnitude in experiment time

STREET: Swedish Tool for Risk/Resource Estimation at EvenTs. Part two, resource assessment – face validity and inter–rater reliability

Objective: To develop a validated and generalized collaborative tool to be utilized by high reliability organizations in order to conduct common resource assessment before major events and mass gatherings. Methods: The Swedish resource and risk estimation guide was used as foundation for the development of the generalized collaborative tool, by three different expert groups, and then analyzed. Analysis of inter-rater reliability was conducted through simulated cases that showed weighted and unweight κ-statistics. Results: The results revealed a mean of unweight κ-value from the three cases of 0.44 and a mean accuracy of 61% of the tool. Conclusions: A better collaboration ability and more accurate resource assessment with acceptable reliability and validity were shown in this study to be used as a foundation for resource assessment before major events/mass-gathering in a simulated environment. However, the result also indicates the challenges of creating measurable values from simulated cases. A study on real events can provide higher reliability but needs, on the other hand, an already developed tool

STREET: Swedish Tool for Risk/Resource Estimation at EvenTs. Part one, risk assessment – face validity and inter–rater reliability

Objective: To develop a validated and generalized high reliability organizations collaborative tool in order to conduct common assessments and information sharing of potential risks during mass-gatherings. Methods: The Swedish resource and risk estimation guide was used as foundation for the development of the generalized collaborative tool, by three different expert groups, and then analyzed. Analysis of inter-rater reliability was conducted through simulated cases that showed weighted and unweight κ-statistics. Results: The results revealed a mean of unweight κ-value from the three cases of 0.37 and a mean accuracy of 62% of the tool. Conclusions: The collaboration tool, “STREET”, showed acceptable reliability and validity to be used as a foundation for high reliability organization collaboration in a simulated environment. However, the lack of reliability in one of the cases highlights the challenges of creating measurable values from simulated cases. A study on real events can provide higher reliability but need, on the other hand, an already developed tool

Rapid NMR Relaxation Rate Measurements Using Optimal Non-Uniform Sampling of Multi-Dimensional Accordion Data Analyzed by a Sparse Reconstruction Method

Nonuniform sampling (NUS) of multidimensional NMR data offers significant time savings while improving spectral resolution or increasing sensitivity per unit time. However, NUS has not been widely used for quantitative analysis because of the nonlinearity of most methods used to model NUS data, which leads to problems in estimating signal intensities, relaxation rate constants, and their error bounds. Here, we present an approach that avoids these limitations by combining accordion spectroscopy and NUS in the indirect dimensions of multidimensional spectra and then applying sparse exponential mode analysis, which is well suited for analyzing accordion-type relaxation data in a NUS context. By evaluating the Cramér-Rao lower bound of the variances of the estimated relaxation rate constants, we achieve a robust benchmark for the underlying reconstruction model. Furthermore, we design NUS schemes optimized with respect to the information theoretical lower bound of the error in the parameters of interest, given a specified number of sampling points. The accordion-NUS method compares favorably with conventional relaxation experiments in that it produces identical results, within error, while shortening the length of the experiment by an order of magnitude. Thus, our approach enables rapid acquisition of NMR relaxation data for optimized use of spectrometer time or accurate measurements on samples of limited lifetime

Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System

The mitochondrial proteome contains proteins from two different genetic systems. Proteins are either synthesized in the cytosol and imported into the different compartments of the organelle or directly produced in the mitochondrial matrix. To ensure proteostasis, proteins are monitored by the mitochondrial quality control system, which will degrade non-native polypeptides. Defective mitochondrial membrane proteins are degraded by membrane-bound AAA-proteases. These proteases are regulated by factors promoting protein turnover or preventing their degradation. Here we determined genetic interactions between the mitoribosome receptors Mrx15 and Mba1 with the quality control system. We show that simultaneous absence of Mrx15 and the regulators of the i-AAA protease Mgr1 and Mgr3 provokes respiratory deficiency. Surprisingly, mutants lacking Mrx15 were more tolerant against proteotoxic stress. Furthermore, yeast cells became hypersensitive against proteotoxic stress upon deletion of MBA1. Contrary to Mrx15, Mba1 cooperates with the regulators of the m-AAA and i-AAA proteases. Taken together, these results suggest that membrane protein insertion and mitochondrial AAA-proteases are functionally coupled, possibly reflecting an early quality control step during mitochondrial protein synthesis