Biology subjects

Davidi, D.

Publications and source records attributed to Davidi, D..

5 recordsLinked to original sources

bioRxiv2023

Systematic exploration of prokaryotic form I rubisco maximal carboxylation rates

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco - the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubiscos carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median 5-fold higher rate. In contrast to prior studies, we find that rubiscos originating from -cyanobacteria exhibit the highest carboxylation rates among form I enzymes ({approx}10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.

systems biology↗

bioRxiv2020

α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

-Synuclein (-Syn) is a protein implicated in the pathogenesis of Parkinsons disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for -Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of -Syn in these mechanisms is currently unclear. Here we have investigated the role of -Syn in membrane trafficking through its association with acidic phosphoinositides (PIPs), such as phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and phosphatidylinositol 3,4-bisphosphate (PI3,4P2). Our results show that -Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor AP2 at clathrin-coated pits. Using endocytosis of transferrin, an indicator of clathrin mediated endocytosis (CME), we find that -Syn involvement in endocytosis is specifically mediated through PI4,5P2 levels. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by -Syn mutations. In accord with their effects on PI4,5P2 levels at the plasma membrane, the PD associated E46K and A53T mutations further enhance SV endocytosis. However, neither A30P mutation, nor Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of -Syn, that interfere with phospholipid binding, affect SV endocytosis. This study provides evidence for a critical involvement of PIPs in -Syn-mediated membrane trafficking. Significance Statement-Synuclein (-Syn) protein is known for its causative role in Parkinsons disease. -Syn is normally involved in mechanisms of membrane trafficking, including endocytosis, exocytosis and synaptic vesicles cycling. However, a certain degree of controversy regarding the exact role of -Syn in these mechanisms persists. Here we show that -Syn acts to increase plasma membrane levels PI4,5P2 and PI3,4P2 to facilitate clathrin mediated and synaptic vesicles endocytosis. Based on the results, we suggest that -Syn interactions with the acidic phosphoinositides facilitate a shift in their homeostasis to support endocytosis.

neuroscience↗

bioRxiv2018

Revisiting tradeoffs in Rubisco kinetic parameters

Rubisco is the most abundant enzyme in the biosphere and one of the best-characterized enzymes. Based on correlations between Rubisco kinetic parameters, it is widely posited that tradeoffs embedded in the catalytic mechanism constrain its specificity and maximum catalytic rate. However, the reasoning that established this view was based on data from {approx}20 organisms. We re-examine these tradeoff models using a dataset from {approx}300 organisms. Most correlations are substantially attenuated, with the inverse relationship between carboxylation kcat and specificity SC/O being a key example. Only one tradeoff model survives in our dataset. In this model, increasing catalytic efficiency (kcat/KM) for carboxylation requires increased catalytic efficiency for the competing oxygenation reaction, evidenced by strong power-law correlation between catalytic efficiencies. Our results imply that Rubisco evolution is constrained primarily by the physicochemical limits of O2/CO2 discrimination, which should reframe efforts to engineer this very central enzyme.

systems biology↗

bioRxiv2018

Systematic assessment of GFP tag position on protein localization and growth fitness in yeast

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on localization and function would promote accurate use of tags in experimental setups. Here we examine the effect of Green Fluorescent Protein (GFP) tagging at either the N or C terminus of budding yeast proteins on localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides step towards broad investigation of protein fusion libraries.\n\nHighlightsO_LIProtein tags are widely used in molecular biology although they may interfere with protein function.\nC_LIO_LIThe subcellular localization of hundreds of proteins in yeast is different when tagged at the N or the C terminus.\nC_LIO_LIA competition based assay enables systematic deciphering of correct tagging terminus for essential proteins.\nC_LIO_LIThe presented approach can be used to derive physiologically relevant tagged libraries.\nC_LI

cell biology↗

bioRxiv2018

Chance and pleiotropy dominate genetic diversity in complex bacterial environments

How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of mutants of Bacillus subtilis glutamate dehydrogenase across 19 different environments - from homogenous single cell populations in liquid media to heterogeneous biofilms, plant roots and soil communities. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. Strikingly, however, 84% of the tested alleles showed opposing fitness effects under different growth conditions (environmental pleiotropy). In biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell communities the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred along biofilm development. Overall, per individual condition, by the combined action of selection, pleiotropy and chance, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated. However, across longer periods and multiple environments, nearly all this diversity would be lost - indeed, considering all environments and conditions we have tested, wild-type is the fittest allele.

microbiology↗