M. K. Mathew

Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins.

Haems are metalloporphyrins that serve as prosthetic groups for various biological processes including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control, metabolic reprogramming and microRNA processing. With a few exceptions, haem is synthesized by a multistep biosynthetic pathway comprising defined intermediates that are highly conserved throughout evolution. Despite our extensive knowledge of haem biosynthesis and degradation, the cellular pathways and molecules that mediate intracellular haem trafficking are unknown. The experimental setback in identifying haem trafficking pathways has been the inability to dissociate the highly regulated cellular synthesis and degradation of haem from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural haem auxotrophs that acquire environmental haem for incorporation into haemoproteins, which have vertebrate orthologues. Here we show, by exploiting this auxotrophy to identify HRG-1 proteins in C. elegans, that these proteins are essential for haem homeostasis and normal development in worms and vertebrates. Depletion of hrg-1, or its paralogue hrg-4, in worms results in the disruption of organismal haem sensing and an abnormal response to haem analogues. HRG-1 and HRG-4 are previously unknown transmembrane proteins, which reside in distinct intracellular compartments. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations and, most strikingly, profound defects in erythropoiesis—phenotypes that are fully rescued by worm HRG-1. Human and worm proteins localize together, and bind and transport haem, thus establishing an evolutionarily conserved function for HRG-1. These findings reveal conserved pathways for cellular haem trafficking in animals that define the model for eukaryotic haem transport. Thus, uncovering the mechanisms of haem transport in C. elegans may provide insights into human disorders of haem metabolism and reveal new drug targets for developing anthelminthics to combat worm infestations.

VDAC is a conserved element of death pathways in plant and animal systems.

Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.

Root apoplastic barriers block Na+ transport to shoots in rice (Oryza sativa L.)

Rice is an important crop that is very sensitive to salinity. However, some varieties differ greatly in this feature, making investigations of salinity tolerance mechanisms possible. The cultivar Pokkali is salinity tolerant and is known to have more extensive hydrophobic barriers in its roots than does IR20, a more sensitive cultivar. These barriers located in the root endodermis and exodermis prevent the direct entry of external fluid into the stele. However, it is known that in the case of rice, these barriers are bypassed by most of the Na+ that enters the shoot. Exposing plants to a moderate stress of 100 mM NaCl resulted in deposition of additional hydrophobic aliphatic suberin in both cultivars. The present study demonstrated that Pokkali roots have a lower permeability to water (measured using a pressure chamber) than those of IR20. Conditioning plants with 100 mM NaCl effectively reduced Na+ accumulation in the shoot and improved survival of the plants when they were subsequently subjected to a lethal stress of 200 mM NaCl. The Na+ accumulated during the conditioning period was rapidly released when the plants were returned to the control medium. It has been suggested that the location of the bypass flow is around young lateral roots, the early development of which disrupts the continuity of the endodermal and exodermal Casparian bands. However, in the present study, the observed increase in lateral root densities during stress in both cultivars did not correlate with bypass flow. Overall the data suggest that in rice roots Na+ bypass flow is reduced by the deposition of apoplastic barriers, leading to improved plant survival under salt stress.

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

Understanding how structure develops during the course of amyloid fibril formation by the prion protein is important for understanding prion diseases. Determining how conformational heterogeneity manifests itself in the fibrillar and pre-fibrillar amyloid aggregates is critical for understanding prion strain phenotypes. In this study, the formation of worm-like amyloid fibrils by the mouse prion protein has been characterized structurally by hydrogen-deuterium exchange coupled to mass spectrometry. The structural cores of these fibrils and of the oligomer on the direct pathway of amyloid fibril formation have been defined, showing how structure develops during fibril formation. The structural core of the oligomer not on the direct pathway has also been defined, allowing the delineation of the structural features that make this off-pathway oligomer incompetent to directly form fibrils. Sequence segments that exhibit multiple local conformations in the three amyloid aggregates have been identified, and the development of structural heterogeneity during fibril formation has been characterized. It is shown that conformational heterogeneity is not restricted to only the C-terminal domain region, which forms the structural core of the aggregates; it manifests itself in the N-terminal domain of the protein as well. Importantly, all three amyloid aggregates are shown to be capable of disrupting lipid membrane structure, pointing to a mechanism by which they may be toxic.

A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast MODULATION OF CYTOSOLIC CALCIUM SIGNATURE AND ACTIVATION OF ADAPTIVE Na+ HOMEOSTASIS

Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca2+ pumps, requires micromolar Ca2+ for growth. In medium containing 100 μm Ca2+, K616 can withstand osmotic stress (750 mm sorbitol) and ionic stress (300 mm KCl) but not hypersodic stress (300 mm NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca2+-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca2+-depleted medium. All stresses tested generated transient elevation of cytosolic Ca2+ in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca2+ in K616. Both the Ca2+ transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na+ predominantly by pumping it out across the plasma membrane, the transformant sequestered Na+ in internal organelles. This sequestration requires the presence of an endomembrane Na+/H+-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na+-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca2+ pump results in the rapid depletion of cytosolic Ca2+ and the activation of an alternate mechanism for surviving saline stress.

Rice cultivars with differing salt tolerance contain similar cation channels in their root cells

Salinity poses a major threat for agriculture worldwide. Rice is one of the major crops where most of the high-yielding cultivars are highly sensitive to salinity. Several studies on the genetic variability across rice cultivars suggest that the activity and composition of root plasma membrane transporters could underlie the observed cultivar-specific salinity tolerance in rice. In the current study, it was found that the salt-tolerant cultivar Pokkali maintains a higher K+/Na+ ratio compared with the salt-sensitive IR20 in roots as well as in shoots. Using Na+ reporter dyes, IR20 root protoplasts showed a much faster Na+ accumulation than Pokkali protoplasts. Membrane potential measurements showed that root cells exposed to Na+ in IR20 depolarized considerably further than those of Pokkali. These results suggest that IR20 has a larger plasma membrane Na+ conductance. To assess whether this could be due to different ion channel properties, root protoplasts from both Pokkali and IR20 rice cultivars were patch-clamped. Voltage-dependent K+ inward rectifiers, K+ outward rectifiers, and voltage-independent, non-selective channels with unitary conductances of around 35, 40, and 10 pS, respectively, were identified. Only the non-selective channel showed significant Na+ permeability. Intriguingly, in both cultivars, the activity of the K+ inward rectifier was drastically down-regulated after plant growth in salt but gating, conductance, and activity of all channel types were very similar for the two cultivars.

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H2O2. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.

Repression of the glucose-inducible outer- membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [¹⁴C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strains ability to utilize aromatics and organic acids over glucose.

Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

Clathrin-dependent endocytosis operates in all cell layers of root, whereas a clathrin-independent pathway operates only in the epidermis, and salinity stress induces an additional clathrin-independent pathway in all cell layers. Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity.

Potassium channel regulator KCNRG regulates surface expression of Shaker-type potassium channels.

Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K(+) channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K(+) currents through human K(+) channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes.