C-terminal truncations of the Saccharomyces cerevisiae PMA1 H+-ATPase have major impacts on protein conformation, trafficking, quality control, and function.

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 Lys(889), removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala(881) (at the end of TM10) to Gly(888) is required for stable folding and PM targeting. (ii) The stretch between Lys(889) and Lys(916), 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 (Glu(917) and Thr(918)) 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.

Drug Sensitivity Testing of Mycobacterium tuberculosis Growing in a Hollow Fiber Bioreactor.

Hollow fiber systems (HFSs) have been widely applied to study pharmacokinetic-pharmacodynamic (PK-PD) relationships in antibiotic research and development. The system comprises a bundle of high-density hollow capillary fibers that conduct a flow of medium with or without drug and an extra-capillary space (ECS) inoculated with the pathogen of interest. The semipermeable membrane of the hollow fibers allows for rapid exchange of small molecule drugs and solutes, while the pathogen is restricted to the ECS. The unique properties of the HFS are (1) the ability to simulate any PK profile within the fibers and ECS, including plasma or site-of-disease PK profiles, (2) the ability to simultaneously input several drugs with different half-lives, (3) the ability to manipulate growth conditions such as medium composition, carbon source, and pH, and (4) the ability to sample in both compartments in order to monitor drug concentrations and bacterial growth kinetics over time. The system is particularly suited for Mycobacterium tuberculosis research in a biosafety level 3 (BSL3) environment since pathogenic bacteria are sequestered in an isolated compartment. The HFS was qualified by the European Medicines Agency for antituberculosis drug development in 2015. Here, we describe the standard procedures used to study the growth kinetics of M. tuberculosis in the HFS and the killing effect of first-line antituberculous drugs applied under simulated human PK conditions. This animal-sparing and economical tool can be applied to optimize dosing schedules that minimize emergence of resistance and to prioritize drug regimens that accelerate sterilization.

CCR3: Shedding new light on a dark problem?

A recent work by Ambati et al. represents a bold step towards a more effective diagnosis and treatment of age-related macular degeneration, with the new evidence showing that CCR3, a chemokine receptor, is an early marker of and potential therapeutic target for choroidal neovascularization development. In the wake of such a novel and significant finding, additional illumination to confirm and consolidate the promise shown by CCR3 will soon follow.

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 Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 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, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 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.

Conformational changes of yeast plasma membrane H(+)-ATPase during activation by glucose: role of threonine-912 in the carboxy-terminal tail.

Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.

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.