Carolyn W. Slayman

Potassium transport in Neurospora: I. Intracellular sodium and potassium concentrations, and cation requirements for growth

Abstract 1. 1. Wild-type Neurospora crassa, growing logarithmically in minimal medium, contains 180 ± 16 mM intracellular K+ and 14 ± 2 mM Na+. It requires K+ but not Na+ for growth. Over a wide range of extracellular K+ concentrations (0.3 to 100 mM), the cells maintain a constant, high level of intracellular K+ (180 mM) and rapid growth occurs. Below 0.3 mM, the internal K+ concentration falls and growth is inhibited. 2. 2. When cells are harvested from logarithmic growth and resuspended in distilled water or in 1 mM sodium azide, they show no significant loss of internal K+ for at least 1 h. Exposure to the polyene antibiotic nystatin (30 μg/ml) or the decapeptide antibiotic tyrocidine (20 μg/ml) causes most of the internal K+ to be lost within 2 min. Both antibiotics are thought to attack the plasma membrane of sensitive organisms. 3. 3. Neurospora exchanges its intracellular K+ quite rapidly for extracellular K+ or Rb+, but only slowly for Na+. 4. 4. These experiments were undertaken to provide a backgroun for microelectrode studies with Neurospora and for the isolation of a K+-transport mutant.

Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H+-ATPase Leads to Glucose-dependent Activation

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H+-ATPase, which is responsible for H+-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5–10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14N/15N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H+-ATPase.

Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage-dependent K+ channels

A degenerate oligonucleotide corresponding to the K+ channel signature sequence (TMTTVGYGD) was used to isolate the genomic and cDNA forms of a new channel gene, AKT3, from Arabidopsis thaliana. The deduced protein sequence has a predicted membrane topography similar to Shaker‐like K+ channels. Three distinct modules comprise the carboxyl‐terminal half: a nucleotide‐binding motif, an ankyrin repeat domain, and a polyglutamate track. Xenopus oocytes injected with cRNA exhibited an inward‐rectifying K+ current, demonstrating that the AKT3 polypeptide is a functional transport protein. Two other Arabidopsis K+ transporters (AKT1 and KAT1) share 60% homology with AKT3; together these proteins constitute a family of plant inward‐rectifying K+ channels.

H+ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.

Measurement of Membrane Potentials in Neurospora

Microelectrodes were used to record intracellularly from the filamentous fungus Neurospora crassa. Under standard conditions membrane potentials averaged 127 mv, inside negative. The potentials were potassium-sensitive, and depended upon the distance of the cells from the growing margin of the colony. In addition, the potentials were quickly reduced to about 30 mv in the presence of low concentrations of sodium azide or the polyene antibiotic nystatin.

Phosphorylation Region of the Yeast Plasma-membrane H+-ATPase ROLE IN PROTEIN FOLDING AND BIOGENESIS

Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6–4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940–7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface.

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: Inhibition by amiloride and its derivatives

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.

Quality Control in the Yeast Secretory Pathway A MISFOLDED PMA1 H+-ATPase REVEALS TWO CHECKPOINTS

The yeast plasma-membrane H+-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H+-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.

Potassium transport in neurospora: II. Measurement of steady-state potassium fluxes

Abstract 1. 1. The radioisotope 42K has been used to measure the steady-state K+ flux in wild-type Neurospora cells, harvested from logarithmic-phase growth and resuspended in buffer. The uptake of 42K by these cells does not depend upon the presence of substrate in the medium, presumably because the cells contain substantial stores of carbohydrate. Uptake is blocked, however, by the respiratory inhibitors sodium azide and 2,4-dinitrophenol. 2. 2. There is competition for uptake between K+ and Rb+. 3. 3. As the extracellular K+ concentration is increased, the steady-state K+ flux saturates. The data have been analyzed in terms of Michaelis-Menten kinetics, and the following constants have been determined: Km = 1 mM; V = 20 mmoles per l cell water per min.

Phosphate transport in Neurospora. Kinetic characterization of a constitutive, low-affinity transport system.

Abstract Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a K 1 2 (at pH 5.8) of 0.123 mM and a Jmax of 1.64 mmoles/l cell water per min. Like the PO43− transport system in yeast, the Neurospora system is stimulated by high intracellular K+. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations. The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the Jmax remains essentially constant but the K 1 2 increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H2PO4− (which would show only a 3-fold increase in apparent K 1 2 over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH− or H+ is assumed to act as a modifier of the transport system, altering its affinity for substrate.