Kaiserslautern - Fachbereich Biologie
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The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program
A building-block model reveals new insights into the biogenesis of yeast mitochondrial ribosomes
(2020)
Most of the mitochondrial proteins in yeast are encoded in the nuclear genome, get synthesized by cytosolic ribosomes and are imported via TOM and TIM23 into the matrix or other subcompartments of mitochondria. The mitochondrial DNA in yeast however also encodes a small set of 8 proteins from which most are hydrophobic membrane proteins and build core components of the OXPHOS complexes. They get synthesized by mitochondrial ribosomes which are descendants of bacterial ribosomes and still have some similarities to them. On the other hand, mitochondrial ribosomes experienced various structural and functional changes during evolution that specialized them for the synthesis of the mitochondrial encoded membrane proteins. The mitoribosome contains mitochondria-specific ribosomal proteins and replaced the bacterial 5S rRNA by mitochondria-specific proteins and rRNA extensions. Furthermore, the mitoribosome is tethered to the inner mitochondrial membrane to facilitate a co-translational insertion of newly synthesized proteins. Thus, also the assembly process of mitoribosomes differs from that of bacteria and is to date not well understood.
Therefore, the biogenesis of mitochondrial ribosomes in yeast should be investigated. To this end, a strain was generated in which the gene of the mitochondrial RNA-polymerase RPO41 is under control of an inducible GAL10-promoter. Since the scaffold of ribosomes is built by ribosomal RNAs, the depletion of the RNA-polymerase subsequently leads to a loss of mitochondrial ribosomes. Reinduction of Rpo41 initiates the assembly of new mitoribosomes, which makes this strain an attractive model to study mitoribosome biogenesis.
Initially, the effects of Rpo41 depletion on cellular and mitochondrial physiology was investigated. Upon Rpo41 depletion, growth on respiratory glycerol medium was inhibited. Furthermore, mitochondrial ribosomal 21S and 15S rRNA was diminished and mitochondrial translation was almost completely absent. Also, mitochondrial DNA was strongly reduced due to the fact that mtDNA replication requires RNA primers that get synthesized by Rpo41.
Next, the effect of reinduction of Rpo41 on mitochondria was tested. Time course experiments showed that mitochondrial translation can partially recover from 48h Rpo41 depletion within a timeframe of 4.5h. Sucrose gradient sedimentation experiments further showed that the mitoribosomal constitution was comparable to wildtype control samples during the time course of 4.5h of reinduction, suggesting that the ribosome assembly is not fundamentally altered in Gal-Rpo41 mitochondria. In addition, the depletion time was found to be critical for recovery of mitochondrial translation and mitochondrial RNA levels. It was observed that after 36h of Rpo41 depletion, the rRNA levels and mitochondrial translation recovered to almost 100%, but only within a time course of 10h.
Finally, mitochondria from Gal-Rpo41 cells isolated after different timepoints of reinduction were used to perform complexome profiling and the assembly of mitochondrial protein complexes was investigated. First, the steady state conditions and the assembly process of mitochondrial respiratory chain complexes were monitored. The individual respiratory chain complexes and the super-complexes of complex III, complex IV and complex V were observed. Furthermore, it was seen that they recovered from Rpo41 depletion within 4.5h of reinduction. Complexome profiles of the mitoribosomal small and large subunit discovered subcomplexes of mitoribosomal proteins that were assumed to form prior to their incorporation into assembly intermediates. The complexome profiles after reinduction indeed showed the formation of these subcomplexes before formation of the fully assembled subunit. In the mitochondrial LSU one subcomplex builds the membrane facing protuberance and a second subcomplex forms the central protuberance. In contrast to the preassembled subcomplexes, proteins that were involved in early assembly steps were exclusively found in the fully assembled subunit. Proteins that assemble at the periphery of the mitoribosome during intermediate and late assembly steps where found in soluble form suggesting a pool of unassembled proteins that supply assembly intermediates with proteins.
Taken together, the findings of this thesis suggest a so far unknow building-block model for mitoribosome assembly in which characteristic structures of the yeast mitochondrial ribosome form preassembled subcomplexes prior to their incorporation into the mitoribosome.
The handling of oxygen sensitive samples and growth of obligate anaerobic organisms
requires the stringent exclusion of oxygen, which is omnipresent in our normal atmospheric
environment. Anaerobic workstations (aka. Glove boxes) enable the handling of
oxygen sensitive samples during complex procedures, or the long-term incubation of
anaerobic organisms. Depending on the application requirements, commercial workstations
can cost up to 60.000 €. Here we present the complete build instructions for a highly
adaptive, Arduino based, anaerobic workstation for microbial cultivation and sample handling,
with features normally found only in high cost commercial solutions. This build can
automatically regulate humidity, H2 levels (as oxygen reductant), log the environmental
data and purge the airlock. It is built as compact as possible to allow it to fit into regular
growth chambers for full environmental control. In our experiments, oxygen levels during
the continuous growth of oxygen producing cyanobacteria, stayed under 0.03 % for 21 days
without needing user intervention. The modular Arduino controller allows for the easy
incorporation of additional regulation parameters, such as CO2 concentration or air pressure.
This paper provides researchers with a low cost, entry level workstation for anaerobic
sample handling with the flexibility to match their specific experimental needs.
Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive—a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions
For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in E. coli and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here we have applied the QconCAT approach to Chlamydomonas reinhardtii for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a Chlamydomonas cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b6/f complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique.
The cytosolic Fe65 adaptor protein family, consisting of Fe65, Fe65L1 and Fe65L2 is involved in many intracellular signaling pathways linking via its three interaction domains a continuously growing list of proteins by facilitating functional interactions. One of the most important binding partners of Fe65 family proteins is the amyloid precursor protein (APP), which plays an important role in Alzheimer Disease.
To gain deeper insights in the function of the ubiquitously expressed Fe65 and the brain enriched Fe65L1, the goal of my study was I) to analyze their putative synaptic function in vivo, II) to examine structural analysis focusing on a putative dimeric complex of Fe65, III) to consider the involvement of Fe65 in mediating LRP1 and APP intracellular trafficking in murine hippocampal neurons. By utilizing several behavioral analyses of Fe65 KO, Fe65L1 KO and Fe65/Fe65L1 DKO mice I could demonstrate that the Fe65 protein family is essential for learning and memory as well as grip strength and locomotor activity. Furthermore, immunohistological as well as protein biochemical analysis revealed that the Fe65 protein family is important for neuromuscular junction formation in the peripheral nervous system, which involves binding of APP and acting downstream of the APP signaling pathway. Via Co-immunoprecipitation analysis I could verify that Fe65 is capable to form dimers ex vivo, which exclusively occur in the cytosol and upon APP expression are shifted to membrane compartments forming trimeric complexes. The influence of the loss of Fe65 and/or Fe65L1 on APP and/or LRP1 transport characteristics in axons could not be verified, possibly conditioned by the compensatory effect of Fe65L2. However, I could demonstrate that LRP1 affects the APP transport independently of Fe65 by shifting APP into slower types of vesicles leading to changed processing and endocytosis of APP.
The outcome of my thesis advanced our understanding of the Fe65 protein family, especially its interplay with APP physiological function in synapse formation and synaptic plasticity.
Biological soil crusts (biocrusts) are a common element of the Queensland (Australia) dry savannah ecosystem and are composed of cyanobacteria, algae, lichens, bryophytes, fungi and heterotrophic bacteria. Here we report how the CO2 gas exchange of the cyanobacteria-dominated biocrust type from Boodjamulla National Park in the north Queensland Gulf Savannah responds to the pronounced climatic seasonality and on their quality as a carbon sink using a semi-automatic cuvette system. The dominant cyanobacteria are the filamentous species Symplocastrum purpurascens together with Scytonema sp. Metabolic activity was recorded between 1 July 2010 and 30 June 2011, during which CO2 exchange was only evident from November 2010 until mid-April 2011, representative of 23.6 % of the 1-year recording period. In November at the onset of the wet season, the first month (November) and the last month (April) of activity had pronounced respiratory loss of CO2. The metabolic active period accounted for 25 % of the wet season and of that period 48.6 % was net photosynthesis (NP) and 51.4 % dark respiration (DR). During the time of NP, net photosynthetic uptake of CO2 during daylight hours was reduced by 32.6 % due to water supersaturation. In total, the biocrust fixed 229.09 mmol CO2 m−2 yr−1, corresponding to an annual carbon gain of 2.75 g m−2 yr−1. Due to malfunction of the automatic cuvette system, data from September and October 2010 together with some days in November and December 2010 could not be analysed for NP and DR. Based on climatic and gas exchange data from November 2010, an estimated loss of 88 mmol CO2 m−2 was found for the 2 months, resulting in corrected annual rates of 143.1 mmol CO2 m−2 yr−1, equivalent to a carbon gain of 1.7 g m−2 yr−1. The bulk of the net photosynthetic activity occurred above a relative humidity of 42 %, indicating a suitable climatic combination of temperature, water availability and light intensity well above 200 µmol photons m−2 s−1 photosynthetic active radiation. The Boodjamulla biocrust exhibited high seasonal variability in CO2 gas exchange pattern, clearly divided into metabolically inactive winter months and active summer months. The metabolic active period commences with a period (of up to 3 months) of carbon loss, likely due to reestablishment of the crust structure and restoration of NP prior to about a 4-month period of net carbon gain. In the Gulf Savannah biocrust system, seasonality over the year investigated showed that only a minority of the year is actually suitable for biocrust growth and thus has a small window for potential contribution to soil organic matter.
Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria
and allow these organisms to absorb in the so-called green gap. They consist of smaller units called
phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound
linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein
lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead
use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the
blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single
PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the
phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function
of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function
are discussed.
The main goal of this work was to characterize the assembly of PE-III and thus the function of the
six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual
lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in
E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE
revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately,
additional chromophorylation could not be observed using the pDuet system. Therefore, in a
second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced
into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli,
no translation was observed, possibly due to incompatible translation initiation between
Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the
cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case
production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was
also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity
and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a
cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and,
based on this, future work could aim at generating an artificial operon using synthetic biology to
achieve efficient translation of all genes.
Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N2 and conduct oxygenic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed. In light of this, the effects of elevated atmospheric CO2 availability on growth, carbon and N2 fixation as well as nodularin production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following:
Biomass and volume specific biological nitrogen fixation (BNF) rates were respectively almost six and 17 fold higher in the aquatic Nodularia species compared to the terrestrial Nostoc species tested, under elevated CO2 conditions.
There was a direct correlation between elevated CO2 and decreased dry weight specific cellular nodularin content in a diazotrophically grown terrestrial Nostoc species, and the aquatic Nodularia species, regardless of nitrogen availability.
Elevated atmospheric CO2 levels were correlated to a reduction in biomass specific BNF rates in non-toxic Nodularia species.
Nodularin producers exhibited stronger stimulation of net photosynthesis rates (NP) and growth (more positive Cohen’s d) and less stimulation of dark respiration and BNF per volume compared to non-nodularin producers under elevated CO2 levels.
This study is the first to provide information on NP and nodularin production under elevated atmospheric CO2 levels for Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions.
The amyloid precursor protein (APP) is a key molecular component of Alzheimer’s disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context
of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm
preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosinebinding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.