MPM motifs of the yeast SKT protein Trk1 can assemble to form a functional K+-translocation system

The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC). The function of these partial Trk1 proteins either alone or in combinations was assessed by expressing the encoding genes in a K⁺-uptake deficient strain lacking also the K-channel Tok1 (trk1,trk2,tok1Δ) and (i) analysing their ability to promote growth in low [K⁺] media and (ii) by ion flux measurements using “microelectrode based ion flux estimation” (MIFE). We found that proteins containing only one or two MPM motifs can interact with each other and assemble with a polypeptide consisting of the rest of the Trk system to form a functional K⁺-translocation system.     

The functional role of conserved acidic residues of the Qcr7 protein of the cytochrome bc(1) complex in Saccharomyces cerevisiae

The 14-kDa Qcr7 protein represents one of the 10 subunits that are components of a functional cytochrome bc(1) complex in Sacharomyces cerevisiae. Previous studies have shown that the N-terminus of the Qcr7 protein may be involved in the assembly of the cytochrome bc(1) complex and its C-terminus by interacting with cytochrome b and QCR8 proteins. It has also been suggested that Qcr7 protein may be involved in proton pumping. The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagenesis and the mutated genes expressed in cells lacking a functional QCR7 gene. Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon sources. Mutants D47G and D47N were respiratory deficient and analysis of complex components by immunoblotting and spectral analysis of cytochrome b suggests defective assembly. Despite being respiratory competent and having normal electron transport rates in broken mitochondria, the mutant D46G had markedly reduced ATP synthesis from electron transport reactions catalyzed by complexes II plus III of the respiratory chain. This suggests that the geometry of proton uptake by the bc(1) complex is disturbed by the mutation in D46.     

Involvement of the penta-EF-hand protein Pef1p in the Ca2+-dependent regulation of COPII subunit assembly in Saccharomyces cerevisiae

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.     

A novel membrane protein capable of binding the Na+/H+ antiporter (Nha1p) enhances the salinity-resistant cell growth of Saccharomyces cerevisiae

The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of approximately 40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Delta cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein.