Functional analysis of the plasma membrane Alr1 and Alr2 proteins in Yeast Saccharomyces cerevisiae

Die Bäckerhefe Saccharomyces cerevisiae besitzt ein sehr raffiniertes System für den Magnesiumtransport in die Zelle, kodiert durch die Gene ALR1 und ALR2. In der vorliegenden Arbeit bestätigt eine Reihe von biochemischen und genetischen Analysen eine funktionelle und strukturelle Homologie von Alr1p und seinem nahen Homolog Alr2p mit der CorA-Mrs2-Alr1 Familie. Während Alr1p als der primäre Mg2+ -Transporter fungiert, trägt das Alr2-Protein unter Standardbedingungen nur wenig zur Mg2+-Aufnahme bei. Die Untersuchungen haben gezeigt, dass der Alr2p vermittelte Mg2+-influx durch die Substitution einer einzelnen Aminosäure von Argxy zu Gluxy in der „loop-Region“ zwischen beiden Transmembranhelices signifikant gesteigert werden konnte. Mittels chemischer „cross-link Studien“ und in vivo „mating-based split-ubuquitin Studien“ konnte sowohl die Ausbildung von Homo- als auch Heterooligomere zwischen Alr1p und Alr2p beobachtet werden. Um die Funktion unterschiedlicher Proteinabschnitte zu analysieren wurden sequenzielle Deletionen sowohl am C- als auch am N-terminus des Proteins durchgeführt und die Funktion bzw. deren Ausfall untersucht. Veränderungen am C-terminalen Ende, nahe der 2. Transmembrandomäne hatten meist einen Funktionsverlust des Proteins zur Folge. Konsekutive Verkürzungen am N-terminus bis zu einem Ausmaß von ca. 270 Aminosäuren hatten keinen Einfluss auf die Funktion des Proteins. Allerdings zeigten sich signifikante Abweichungen in der Proteinstabilität und Lokalisation dieser Isomere. Deletionen von mehr als 270 aa führten zu einem totalen Funktionsverlust. Proteine mit einer Deletion im Abschnitt zwischen E271 und Q318 wurden nicht mehr zur Plasmamembran transloziert und wiesen eine sehr geringe, von Magnesium unabhängige, Stabilität auf. Mit größter Wahrscheinlichkeit werden diese defekten Proteine durch „Sorting Prozesse“ des Endoplasmatischen Reticulums bzw. des Golgiapparates erkannt und dem proteosomalen Abbau zugeführt.Proteins Alr1 and Alr2 of Saccharomyces cerevisiae encode a very sophisticated system to take up magnesium, one of the most abundant cations, into the cell. In this thesis a series of biochemical and genetic analyses support the functional and structural homology of Alr1p and its close homolog protein Alr2 to the CorA-Mrs2-Alr1 family. Whereas Alr1p acts as the main Mg2+-transporter, Alr2p contributes poorly to Mg2+-uptake. Substitution of a single arginine with a glutamic acid residue in the loop between the two TM domains greatly improves its activity. Both, Alr1 and Alr2 proteins are shown to form homo- and hetero-oligomers. Furthermore, this assay also showed that the orientation of Alr proteins is consistent with CorA, where both N-terminal and C-terminal ends are exposed to the cell interior. Successive truncations were constructed from N- and C-terminal tails of Alr1p to dissect the role in protein function. The analysis of carboxyl truncations affirmed the importance of this region for Alr1p Mg2+ transport activity and its critical impact on channel formation. In contrast, consecutive shortening of N-terminal domain of Alr1p accounted for striking abnormalities concerning protein stability and subcellular localization. Further analysis using short amino acid intragenic deletions led to the identification of a sequence element of about 50 amino acids in the amino terminal tail essential for protein translocation and stability. Proteins missing this region were blocked in translocation to the plasma membrane and undergo Mg2+ independent degradation, most likely via sorting mechanisms of the ER and subsequential proteasomal decay

Halophilanema prolata n. gen., n. sp. (Nematoda: Allantonematidae), a parasite of the intertidal bug, Saldula laticollis (Reuter)(Hemiptera: Saldidae) on the Oregon coast

This is the publisher’s final pdf. The published article is copyrighted by BioMed Central and can be found at: http://www.biomedcentral.com/.Background: It is rare to find terrestrial nematode lineages parasitizing arthropods inhabiting the intertidal or\ud littoral zone of the oceans. During an ecological study along the Oregon dunes, an allantonematid nematode\ud (Tylenchomorpha: Allantonematidae) was discovered parasitizing the intertidal shore bug, Saldula laticollis (Reuter)\ud (Hemiptera: Saldidae). This shore bug is adapted to an intertidal environment and can survive short periods of\ud submergence during high tides. The present study describes the nematode parasite and discusses aspects of its\ud development, ecology and evolution.\ud Methods: Adults and last instar nymphs of S. laticollis (Hemiptera: Saldidae) were collected from the high intertidal\ud zone among clumps of Juncus L. (Juncaceae) plants at Waldport, Oregon on October 3, 2011. The bugs were\ud dissected in 1% saline solution and the nematodes killed in 1% Ringers solution and immediately fixed in 5%\ud formalin (at 20°C). Third stage juveniles removed from infected hosts were maintained in 1% saline solution until\ud they matured to the adult stage, molted and mated.\ud Results: Halophilanema prolata n. gen., n. sp. (Nematoda: Allantonematidae) is described from last instar nymphs\ud and adults of the intertidal bug, Saldula laticollis on the Oregon coast. The new genus can be distinguished from\ud other genera in the Allantonematidae by a stylet lacking basal knobs in both sexes, an excretory pore located\ud behind the nerve ring, ribbed spicules, a gubernaculum, the absence of a bursa and the elongate-tubular shape of\ud the ovoviviparous parasitic females. Studies of the organogenesis of Halophilanema showed development to third\ud stage juveniles in the uterus of parasitic females. Maturation to the free-living adults and mating occurred in the\ud environment. The incidence of infection of S. laticollis ranged from 0% to 85% depending on the microhabitat in\ud the intertidal zone.\ud Conclusions: Based on the habitat and morphological characters, it is proposed that Halophilanema adapted a\ud parasitic existence fairly recently, evolutionarily speaking. It was probably a free-living intertidal or shore nematode\ud that fed on microorganisms, especially fungi, in the intertidal habitat and became parasitic after saldids entered the\ud environment. Halophilanema represents the first described nematode parasite of an intertidal insect

Mrs2p Forms a High Conductance Mg2+ Selective Channel in Mitochondria

Members of the CorA-Mrs2-Alr1 superfamily of Mg2+ transporters are ubiquitous among pro- and eukaryotes. The crystal structure of a bacterial CorA protein has recently been solved, but the mode of ion transport of this protein family remained obscure. Using single channel patch clamping we unequivocally show here that the mitochondrial Mrs2 protein forms a Mg2+-selective channel of high conductance (155 pS). It has an open probability of ∼60% in the absence of Mg2+ at the matrix site, which decreases to ∼20% in its presence. With a lower conductance (∼45 pS) the Mrs2 channel is also permeable for Ni2+, whereas no permeability has been observed for either Ca2+, Mn2+, or Co2+. Mutational changes in key domains of Mrs2p are shown either to abolish its Mg2+ transport or to change its characteristics toward more open and partly deregulated states. We conclude that Mrs2p forms a high conductance Mg2+ selective channel that controls Mg2+ influx into mitochondria by an intrinsic negative feedback mechanism

Regulation of Alr1 Mg Transporter Activity by Intracellular Magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms