The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4.

Lpe10p modulates the activity of the Mrs2p-based yeast mitochondrial Mg2+ channel.

Saccharomyces cerevisiae Lpe10p is a homologue of the Mg(2+)-channel-forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2Delta/lpe10Delta double disruption, indicating that these two proteins cannot substitute for each other.

Determination of yeast mitochondrial KHE activity, osmotic swelling and mitophagy.

The mitochondrial K(+)/H(+) exchanger (KHE) is a key regulator of mitochondrial K(+), the most abundant cellular cation, and thus for volume control of the organelle. Downregulation of the mitochondrial KHE results in osmotic swelling and autophagic degradation of the organelle. This chapter describes methods to shut-off expression of Mdm38p, an essential factor of the mitochondrial KHE, and to observe the cellular consequences thereof, in particular changes in KHE activity and morphogenetic changes of mitochondria by applying new techniques developed in our laboratories.

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane.

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe(2+) homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe(2+) utilization or transport under iron-limiting conditions.

Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL(5)KLK (KLK).

We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane.

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose.

Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.

SLC41A1 is a novel mammalian Mg2+ carrier.

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments.

Mutational analysis of functional domains in Mrs2p, the mitochondrial Mg2+ channel protein of Saccharomyces cerevisiae.

The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants.

Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins.

YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K+ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K+- and H+-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K+ and H+ transport across the yeast inner mitochondrial membrane.

Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica.

The Mg2+ fluorescent dye mag-fura 2, entrapped in cells or organelles, has frequently been used for dual excitation ratio-metric determinations of free ionic Mg2+ concentrations in eukaryotic, mostly mammalian cells. Here we report its successful application to measure free Mg2+ concentrations ([Mg2+]i) in Salmonella enterica cells. When kept in nominally Mg2+ free buffer (resting conditions), the [Mg2+]i of wild-type cells has been determined to be 0.

The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome.

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane.