A network-based modeling framework reveals the core signal transduction network underlying high carbon dioxide-induced stomatal closure in guard cells.

Stomata are pores on plant aerial surfaces, each bordered by a pair of guard cells. They control gas exchange vital for plant survival. Understanding how guard cells respond to environmental signals such as atmospheric carbon dioxide (CO2) levels is not only insightful to fundamental biology but also relevant to real-world issues of crop productivity under global climate change. In the past decade, multiple important signaling elements for stomatal closure induced by elevated CO2 have been identified. Yet, there is no comprehensive understanding of high CO2-induced stomatal closure. In this work, we assemble a cellular signaling network underlying high CO2-induced stomatal closure by integrating evidence from a comprehensive literature analysis. We further construct a Boolean dynamic model of the network, which allows in silico simulation of the stomatal closure response to high CO2 in wild-type Arabidopsis thaliana plants and in cases of pharmacological or genetic manipulation of network nodes. Our model has a 91% accuracy in capturing known experimental observations. We perform network-based logical analysis and reveal a feedback core of the network, which dictates cellular decisions in closure response to high CO2. Based on these analyses, we predict and experimentally confirm that applying nitric oxide (NO) induces stomatal closure in ambient CO2 and causes hypersensitivity to elevated CO2. Moreover, we predict a negative regulatory relationship between NO and the protein phosphatase ABI2 and find experimentally that NO inhibits ABI2 phosphatase activity. The experimental validation of these model predictions demonstrates the effectiveness of network-based modeling and highlights the decision-making role of the feedback core of the network in signal transduction. We further explore the model's potential in predicting targets of signaling elements not yet connected to the CO2 network. Our combination of network science, in silico model simulation, and experimental assays demonstrates an effective interdisciplinary approach to understanding system-level biology.

The yeast protein Gdt1p transports Mn2+ ions and thereby regulates manganese homeostasis in the Golgi.

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn2+ homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis Using Mn2+-induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn2+ influx, in addition to its previously reported regulation of Ca2+ influx. The estimated Km values of Gdt1p of 15.6 ± 2.6 µm for Ca2+ and 83.2 ± 9.8 µm for Mn2+ indicated that Gdt1p has a higher affinity for Ca2+ than for Mn2+ In yeast cells, we found that Gdt1p is involved in the resistance to high Mn2+ concentration and controls total Mn2+ stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn2+-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn2+ concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn2+ homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation.

Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca(2+) exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca(2+) uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca(2+)/H(+) antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn(2+) to the cells.

Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na(+)/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

Functional expression, purification and reconstitution of the recombinant phosphate transporter Pho89 of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae high-affinity phosphate transporter Pho89 is a member of the inorganic phosphate (Pi) transporter (PiT) family, and shares significant homology with the type III Na(+)/Pi symporters, hPit1 and hPit2. Currently, detailed biochemical and biophysical analyses of Pho89 to better understand its transport mechanisms are limited, owing to the lack of purified Pho89 in an active form. In the present study, we expressed functional Pho89 in the cell membrane of Pichia pastoris, solubilized it in Triton X-100 and foscholine-12, and purified it by immobilized nickel affinity chromatography combined with size exclusion chromatography. The protein eluted as an oligomer on the gel filtration column, and SDS/PAGE followed by western blotting analysis revealed that the protein appeared as bands of approximately 63, 140 and 520 kDa, corresponding to the monomeric, dimeric and oligomeric masses of the protein, respectively. Proteoliposomes containing purified and reconstituted Pho89 showed Na(+)-dependent Pi transport activity driven by an artificially imposed electrochemical Na(+) gradient. This implies that Pho89 operates as a symporter. Moreover, its activity is sensitive to the Na(+) ionophore monensin. To our knowledge, this study represents the first report on the functional reconstitution of a Pi-coupled PiT family member.

The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae.

BACKGROUND: The Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation. RESULTS: By employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant. CONCLUSIONS: The extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.