Kenneth E. Allen

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.

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.

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.

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H+-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

Effects of C-terminal Truncations on Trafficking of the Yeast Plasma Membrane H+-ATPase

Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1Δ452 to 348 min in Pma1Δ901, but even the longest construct was considerably less stable than wild-type ATPase (t½ = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1Δ901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1Δ901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.

Stalk Segment 4 of the Yeast Plasma Membrane H+-ATPase MUTATIONAL EVIDENCE FOR A ROLE IN THE E1-E2 CONFORMATIONAL CHANGE

In the P2-type ATPases, there is growing evidence that four α-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P2-ATPases. When site-directed mutants in this region of the yeast plasma membrane H+-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC50 values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K m for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E2 conformation of the ATPase toward the E1conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an α-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane.

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

Background: Fluoride is broadly toxic, and organisms use fluoride export (FEX) proteins to expel it. Results: FEX is a constitutively expressed fluoride channel, and mutations to the C- and N-terminal domains have asymmetric effects. Conclusion: Protection from fluoride is constantly needed, and a positive residue in the membrane is required. Significance: Understanding FEX furthers our knowledge of fluoride resistance mechanisms. Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm.

C-Terminal Truncations of the Saccharomyces cerevisiae PMA1 H+-ATPase Have Major Impacts on Protein Conformation, Trafficking, Quality Control, and Function

ABSTRACT The C-terminal tail of yeast plasma membrane (PM) H+-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys889, removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala881 (at the end of TM10) to Gly888 is required for stable folding and PM targeting. (ii) The stretch between Lys889 and Lys916, a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu917 and Thr918) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H+-ATPase.

Coordination of K+ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

ABSTRACT Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K+] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K+ and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1, viz., 10- to 50-fold larger than that in K+- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K+ stress by means of progressively increasing transporter affinity for extracellular K+. The underlying observations are (iv) that K+ influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K+ is progressively depleted below 0.05 mM and (v) that K+ influx via Hak1p remains at ∼3 mM/h when extracellular K+ is depleted below 0.1 mM.