P. Supply

Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene

In plants, the proton pump-ATPase (H+-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H+-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H+-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 μM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, a low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions.

Regulation of the expression of the H+-ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae

A peak of plasma membrane H(+)-ATPase activity during exponential growth is correlated with the expression of the PMA1 gene as monitored by measurements of the beta-galactosidase activity from a PMA1-lacZ fusion. This peak of activity is also correlated to the content of the H(+)-ATPase protein in yeast plasma membrane as shown by quantitative immunodetection. The PMA2-lacZ fusion assay indicates that the expression of the PMA2 gene is activated somewhat later during exponential phase but under all circumstances its activity remains at least 500-fold lower than that of the PMA1-lacZ fusion. A slight but significant stimulation of ATPase activity by low concentrations of octanoic acid coincides with a decrease in the PMA1 gene expression. It is concluded that octanoic acid stimulates de PMA1 ATPase activity by posttranslational mechanisms.

Review: subcellular traffic of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae.

Phosphorylated catalytic intermediate.45 Members of this protein super- family are found in all species from mycoplasma to plants and animals. H+-ATPase transforms the chemical energy contained in the ubiquitous bio- chemical energy mediator, ATP, into the chemio- osmotic energy of a proton transmembrane gradient, which is utilized by secondary transport systems.36 The existence of an electrogenic proton ATPase, which drives transport of metabolites across the fungal plasma membrane, was gradually estab- lished: first by electrophysiological and physiologi- cal observations of coupled proton and metabolite fluxes,

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The plasma membrane H+‐ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+‐ATPase‐depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme’s carboxy terminus. Functional chimerical H+‐ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site‐directed mutagenesis of the H+‐ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H+‐ATPase. The reverse Ala‐598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H+‐ATPase substituted with alanine were enlarged and grew more slowly than wild‐type cells. ATPase activity showed a more alkaline pH optimum, lower Km values for MgATP and decreased Vmax compared with wild‐type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose‐starved cells, indicating that the S. pombe H+‐ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.