EXTINCTION SPACE—A METHOD FOR THE QUANTIFICATION AND CLASSIFICATION OF CHANGES IN MORPHOSPACE ACROSS EXTINCTION BOUNDARIES

Three main modes of extinction are responsible for reductions in morphological disparity: (1) random (caused by a nonselective extinction event); (2) marginal (a symmetric, selective extinction event trimming the margin of morphospace); and (3) lateral (an asymmetric, selective extinction event eliminating one side of the morphospace). These three types of extinction event can be distinguished from one another by comparing changes in three measures of morphospace occupation: (1) the sum of range along the main axes; (2) the sum of variance; and (3) the position of the centroid. Computer simulations of various extinction events demonstrate that the pre‐extinction distribution of taxa (random or normal) in the morphospace has little influence on the quantification of disparity changes, whereas the modes of the extinction events play the major role. Together, the three disparity metrics define an “extinction‐space” in which different extinction events can be directly compared with one another. Application of this method to selected extinction events (Frasnian‐Famennian, Devonian‐Carboniferous, and Permian‐Triassic) of the Ammonoidea demonstrate the similarity of the Devonian events (selective extinctions) but the striking difference from the end‐Permian event (nonselective extinction). These events differ in their mode of extinction despite decreases in taxonomic diversity of similar magnitude.     

Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.     

The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase

A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.     

Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways

Metabolic adaptation of Saccharomyces cerevisiae cells from a nonfermentable carbon source to glucose induces selective, rapid breakdown of the gluconeogenetic key enzyme fructose-1,6-bisphosphatase (FBPase), a process called catabolite degradation. Herein, we identify eight novel GID genes required for proteasome-dependent catabolite degradation of FBPase. Four yeast proteins contain the CTLH domain of unknown function. All of them are Gid proteins. The site of catabolite degradation has been controversial until now. Two FBPase degradation pathways have been described, one dependent on the cytosolic ubiquitin-proteasome machinery, and the other dependent on vacuolar proteolysis. Interestingly, three of the novel Gid proteins involved in ubiquitin-proteasome-dependent degradation have also been reported by others to affect the vacuolar degradation pathway. As shown herein, additional genes suggested to be essential for vacuolar degradation are unnecessary for proteasome-dependent degradation. These data raise the question as to whether two FBPase degradation pathways exist that share components. Detailed characterization of Gid2p demonstrates that it is part of a soluble, cytosolic protein complex of at least 600 kDa. Gid2p is necessary for FBPase ubiquitination. Our studies have not revealed any involvement of vesicular intermediates in proteasome-dependent FBPase degradation. The influence of Ubp14p, a deubiquitinating enzyme, on proteasome-dependent catabolite degradation was further uncovered.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.