Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin–proteasome system

Peroxisome autophagy, also known as pexophagy, describes the wholesale degradation of peroxisomes via the vacuole, when organelles become damaged or redundant. In the methylotrophic yeast Hansenula polymorpha, pexophagy is stimulated when cells growing on methanol are exposed to excess glucose. Degradation of the peroxisomal membrane protein Pex3p, a process that does not involve the vacuole, was shown to trigger pexophagy. In this contribution, we have characterised pexophagy-associated Pex3p degradation further. We show that Pex3p breakdown depends on ubiquitin and confirm that Pex3p is a target for ubiquitination. Furthermore, we identify a role for the peroxisomal E3 ligases Pex2p and Pex10p in Pex3p degradation, suggesting the existence of a ubiquitin-dependent pathway involved in removing proteins from the peroxisomal membrane.     

Analysis of the β-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Δ³,Δ²-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10–25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through β-oxidation was more severely reduced in mutants devoid of Δ³,Δ²-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Δ³,Δ²-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Impairment of peroxisomal β-oxidation system by endotoxin treatment

It is now clear that peroxisomes play a crucial role in many cellular processes, including the -oxidation of very long chain fatty acids. Recently, mammalian peroxisomes have been shown to contain the antioxidant enzymes, superoxide dismutase and glutathione peroxidase, in addition to catalase. The presence of these enzymes in peroxisomes suggests that peroxisomes undergo oxidative stress in normal and disease states. As an indicator of the potential impact of an oxidative stress on peroxisomal functions, we evaluated the effect of endotoxin exposure on the -oxidation enzyme system in rat liver. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment decreased the -oxidation of lignoceric acid to 56% of control values (p<0.01). The specific activity of the rate limiting enzyme in the system, acyl-CoA oxidase, was decreased to 73% of control values (p<0.05). Immunoblot analysis revealed a 25% decrease in the 21KD subunit of the acyl-CoA oxidase protein. In contrast, the protein levels of the other enzymes in the pathway, trifunctional protein and 3-ketoacyl-CoA thiolase, were increased by 10 and 15%, respectively. These findings suggest that impairment of -oxidation of lignoceric acid by endotoxin treatment is due primarily to a reduction in the activity and protein level of the key enzyme, acyl-CoA oxidase. Oxidative stresses such as endotoxin exposure may have deleterious effects on important peroxisomal functions, such as -oxidation of very long chain fatty acids.     

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ?foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ?scdA?echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ?scdA?echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.     

Incorporation of acetyl-CoA generated from peroxisomal β-oxidation into ethanolamine plasmalogen of rat liver

We have reported that peroxisomal β-oxidation has an anabolic function, supplying acetyl-CoA for biosynthesis of bile acids and phospholipids. Here we deal with its role in the biosynthesis of the subclasses of ethanolamine- and choline-containing phosphoglycerides (EPG, CPG, respectively). Rats were fed for 2 weeks on chow containing 0.25% clofibrate, which inhibits cholesterol and bile acid biosyntheses, but stimulates peroxisomal β-oxidation. [1-¹⁴C]Lignoceric acid, which is exclusively degraded by peroxisomal β-oxidation to acetyl-CoA, was intravenously injected, and 3 h later the rats were killed. The EPG-rich and CPG-rich fractions were prepared from the liver. When they were treated with phospholipase A₂, the radioactivity was predominantly recovered in the 1-radyl group. The radioactivity in EPG was easily dissociated with HCl vapor, and the lipid containing radioactivity was found to be a fatty aldehyde mixture consisting of steary aldehyde (approx. 58%) palmityl aldehyde (approx. 40%) and oleyl aldehyde (approx. 2%). Thus, in the case of EPG, acetyl-CoA from peroxisomal β-oxidation is incorporated mainly into the 1-alkenyl group of ethanolamine plasmalogen. The radioactivity in CPG, however, was found in fatty alcohol (formed from fatty acid), but not in alkylglycerol after reduction of the fraction with Vitride. Thus, in the case of CPG, acetyl-CoA from peroxisomal β-oxidation is exclusively incorporated into the 1-acyl group of diacyl glycerophosphocholine, but not into the 1-alkyl group. The above results were supported by the results of phospholipase C treatment. The above data indicate that peroxisomal β-oxidation plays a role in supplying acetyl-CoA for 1-alkenyl group of plasmalogen-type phospholipid, but this channel may open only to synthesis of EPG, and almost not to CPG.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

Postfixation detergent treatment liberates the membrane modelling protein Pex11β from peroxisomal membranes

Pex11 proteins are involved in membrane remodelling processes of peroxisomes, and are key components of peroxisomal division and proliferation. In mammals, three Pex11 isoforms, Pex11α, Pex11β, and Pex11γ exist. Here we demonstrate that Pex11β, but not Pex11α or Pex11γ, is almost exclusively extracted from peroxisomal membranes of paraformaldehyde-fixed cells by permeabilisation with the non-ionic detergent Triton X-100. This results in diminished detection of Myc-Pex11β in immunofluorescence preparations and appearance of the protein in the Triton X-100 extract. To our knowledge, Pex11β is the first peroxisomal membrane protein showing such a peculiar behaviour. Loss of Pex11β can be avoided by permeabilisation with digitonin, the addition of glutaraldehyde to the fixative, or the expression of a Pex11 fusion protein with a larger protein tag (e.g. YFP). Our observations further point to different functions and biochemical properties of the Pex11 isoforms within the peroxisomal membrane and during peroxisome proliferation.     

Immunocytochemical detection of peroxisomal β-oxidation enzymes in cryostat and paraffin sections of human post mortem liver

Summary The immunocytochemical visualization of the peroxisomal -oxidation enzymes was investigated in three human post mortem liver samples. Acyl-CoA oxidase, bifunctional protein and 3-oxoacyl-CoA thiolase remained immunocytochemically detectable 30, 55 and 72 h after death. Peroxisomes in the parenchymal cells were clearly visualized for light microscopy (paraffin and cryostat sections), using protein A-gold in combination with silver enhancement. In two samples catalase activity became very weak, but catalase antigenicity was well preserved. The findings prove the diagnOstic value of post mortem samples, even after extreme conditions of tissue conservation. The technique of immunocytochemical staining for the peroxisomal -oxidation enzymes on unmounted cryostat sections has not been reported previously. This method allows a quick diagnOsis of biopsies from patients suspected of peroxisomal disorders.     

The unusual extended C-terminal helix of the peroxisomal α/β-hydrolase Lpx1 is involved in dimer contacts but dispensable for dimerization

The yeast peroxisomal hydrolase Lpx1 belongs to the α/β-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9 ?. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand β6 of the canonical α/β-hydrolase fold. A four-helix cap domain covers the active site. The interface between the α/β-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The α/β-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000 ?2. The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer.     

The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Effect of cortisone on the developmental pattern of the neutral and the acid β-galactosidases of the small intestine of the rat

1. The developmental pattern and effect of cortisone on acid beta-galactosidase and neutral beta-galactosidase were studied in postnatal rats by a recently proposed method for their independent determination. 2. After birth the acid beta-galactosidase activity increases in the ileum, whereas it decreases slightly in the jejunum. On day 16 after birth the activity in the ileum decreases and in 20-day-old rats activity in both parts of the intestine decreases to adult values. In suckling animals the activity in the ileum exceeds the jejunal activity severalfold and in adult animals the activity in the jejunum is slightly higher than that in the ileum. 3. Neutral beta-galactosidase activity is high after birth and decreases in both jejunum and ileum after day 20 after birth. In 12-20-day-old rats activity in both parts is essentially the same, but in adult animals jejunal activity exceeds ileal activity four-to five-fold. 4. Cortisone (0.5, 2.0 or 5.0mg/100g body wt. daily for 4 days) does not influence the activity of either enzyme in 60-day-old rats. Acid beta-galactosidase activity is decreased after cortisone treatment in 8-, 12-, 16-and 18-day-old rats, with sensitivity to cortisone increasing with the approach of weaning. No effect of cortisone on acid beta-galactosidase is seen in 8-day-old rats. Neutral beta-galactosidase activity is increased in the ileum of 8-, 12-, 16- and 18-day old rats, but only in the jejunum of 8-and 12-day-old rats.     

Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid β-oxidation

The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.     

Gut microbial metabolites of linoleic acid are metabolized by accelerated peroxisomal β-oxidation in mammalian cells

Microorganisms in animal gut produce unusual fatty acids from the ingested diet. Two types of hydroxy fatty acids (HFAs), 10-hydroxy-cis-12-octadecenoic acid (HYA) and 10-hydroxy-octadecanoic acid (HYB), are linoleic acid (LA) metabolites produced by Lactobacillus plantarum. In this study, we investigated the metabolism of these HFAs in mammalian cells. When Chinese hamster ovary (CHO) cells were cultured with HYA, approximately 50% of the supplemented HYA disappeared from the dish within 24 h. On the other hand, the amount of HYA that disappeared from the dish of peroxisome (PEX)-deficient CHO cells was lower than 20%. Significant amounts of C2– and C4-chain-shortened metabolites of HYA were detected in culture medium of HYA-supplemented CHO cells, but not in medium of PEX-deficient cells. These results suggested that peroxisomal β-oxidation is involved in the disappearance of HYA. The PEX-dependent disappearance was observed in the experiment with HYB, but not with LA. We also found that HYA treatment up-regulates peroxisomal β-oxidation activity of human gastric MKN74 cells and intestinal Caco-2 cells. These results indicate a possibility that HFAs produced from gut bacteria affect lipid metabolism of host via modulation of peroxisomal β-oxidation activity.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Phylogeography of the harlequin beetle-riding pseudoscorpion and the rise of the Isthmus of Panamá

Molecular and geological evidence indicates that the emergence of the Isthmus of Panamá influenced the historical biogeography of the Neotropics in a complex, staggered manner dating back at least 9 Myr bp. To assess the influence of Isthmus formation on the biogeography of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides, we analysed mitochondrial COI sequence data from 71 individuals from 13 locations in Panamá and northern South America. Parsimony and likelihood-based phylogenies identified deep divergence between South American and Panamanian clades. In contrast to low haplotype diversity in South America, the Panamanian Cordylochernes clade is comprised of three highly divergent lineages: one clade consisting predominantly of individuals from central Panamá (PAN A), and two sister clades (PAN B1 and PAN B2) of western Panamanian pseudoscorpions. Breeding experiments demonstrated a strictly maternal mode of inheritance, indicating that our analyses were not confounded by nuclear-mitochondrial pseudogenes. Haplotype diversity is striking in western Atlantic Panamá, where all three Panamanian clades can occur in a single host tree. This sympatry points to the existence of a cryptic species hybrid zone in western Panamá, a conclusion supported by interclade crosses and coalescence-based migration rates. Molecular clock estimates yield a divergence time of approximately 3 Myr between the central and western Panamanian clades. Taken together, these results are consistent with a recent model in which a transitory proto-Isthmus enabled an early wave of colonization out of South America at the close of the Miocene, followed by sea level rise, inundation of the terrestrial corridor and then a second wave of colonization that occurred when the Isthmus was completed approximately 3 Myr bp.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

Did the evolution of the phytoplankton fuel the diversification of the marine biosphere?

We discuss the possible links between the fossil record of marine biodiversity, nutrient availability and primary productivity. The parallelism of the fossil records of marine phytoplankton and faunal biodiversity implicates the quantity (primary productivity) and quality (stoichiometry) of phytoplankton as being critical to the diversification of the marine biosphere through the Phanerozoic. The relatively subdued marine biodiversity of the Palaeozoic corresponds to a time of relatively low macronutrient availability and poor food quality of the phytoplankton as opposed to the diversification of the Modern Fauna through the Mesozoic–Cenozoic. Increasing nutrient runoff to the oceans through the Phanerozoic resulted from orogeny, the emplacement of Large Igneous Provinces (LIPs), the evolution of deep-rooting forests and the appearance of more easily decomposable terrestrial organic matter that enhanced weathering. Positive feedback by bioturbation of an expanding benthos played a critical role in evolving biogeochemical cycles by linking the oxidation of dead organic matter and the recycling of nutrients back to the water column where they could be re-utilized. We assess our conclusions against a recently published biogeochemical model for geological time-scales. Major peaks of marine diversity often occur near rising or peak fluxes of silica, phosphorus and dissolved reactive oceanic phosphorus; either major or minor ⁸⁷Sr/⁸⁶Sr peaks; and frequently in the vicinity of major (Circum-Atlantic Magmatic Province) and minor volcanic events, some of which are associated with Oceanic Anoxic Events. These processes appear to be scale-dependent in that they lie on a continuum between biodiversification on macroevolutionary scales of geological time and mass extinction.