Publications by authors named "Airat Valiakhmetov"

4 Publications

  • Page 1 of 1

Phosphate efflux as a test of plasma membrane leakage in cells.

Can J Microbiol 2021 Mar 10;67(3):226-230. Epub 2020 Sep 10.

FRC Pushchino Center for Biological Research, Russian Academy of Sciences, Skryabin Institute of Biochemistry and Physiology of Microorganisms, pr. Nauki 5, Pushchino, 142290 Russia.

Plasma membrane integrity is a key to cell viability. Currently, the main approach to assessing plasma membrane integrity is the detection of penetration of special dyes, such as trypan blue and propidium iodide, into the cells. However, this method needs expensive equipment: a fluorescent microscope or a flow cytometer. Besides, staining with propidium iodide occasionally gives false-positive results. Here, we suggest the phosphate (Pi) leakage assay as an approach to assess the increase in permeability of the plasma membrane of yeast cells. We studied the dependence of phosphate efflux and uptake into cells on the composition of the incubation medium, time, and ambient pH. The difference in optimal conditions for these processes suggests that Pi efflux is not conducted by the Pi uptake system. The Pi efflux in water correlated with the proportion of cells stained with propidium iodide. This indicated that Pi efflux is associated with cytoplasmic membrane disruption in a portion of the yeast cell population. The assay of Pi efflux was used to evaluate membrane disruption in cells treated with some heavy metal ions and detergents.
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http://dx.doi.org/10.1139/cjm-2020-0040DOI Listing
March 2021

The Reduced Level of Inorganic Polyphosphate Mobilizes Antioxidant and Manganese-Resistance Systems in .

Cells 2019 05 15;8(5). Epub 2019 May 15.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Center for Biological Research of the Russian Academy of Sciences, pr. Nauki 5, Pushchino 142290, Russia.

Inorganic polyphosphate (polyP) is crucial for adaptive reactions and stress response in microorganisms. A convenient model to study the role of polyP in yeast is the strain CRN/PPN1 that overexpresses polyphosphatase Ppn1 with stably decreased polyphosphate level. In this study, we combined the whole-transcriptome sequencing, fluorescence microscopy, and polyP quantification to characterize the CRN/PPN1 response to manganese and oxidative stresses. CRN/PPN1 exhibits enhanced resistance to manganese and peroxide due to its pre-adaptive state observed in normal conditions. The pre-adaptive state is characterized by up-regulated genes involved in response to an external stimulus, plasma membrane organization, and oxidation/reduction. The transcriptome-wide data allowed the identification of particular genes crucial for overcoming the manganese excess. The key gene responsible for manganese resistance is encoding a low-affinity manganese transporter: Strong down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, , the top up-regulated gene in CRN/PPN1, is also strongly up-regulated in the manganese-adapted parent strain. Phm7 is an unannotated protein, but manganese adaptation is significantly impaired in Δ, thus suggesting its essential function in manganese or phosphate transport.
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http://dx.doi.org/10.3390/cells8050461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562782PMC
May 2019

Activation of H+-ATPase of the plasma membrane of Saccharomyces cerevisiae by glucose: the role of sphingolipid and lateral enzyme mobility.

PLoS One 2012 16;7(2):e30966. Epub 2012 Feb 16.

Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

Activation of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H(+)-ATPase oligomerization for the activation of H(+)-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H(+)-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H(+)-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H(+)-ATPase clusters in the PM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030966PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281057PMC
August 2012

Molecular architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase.

J Biol Chem 2003 Feb 11;278(8):6330-6. Epub 2002 Dec 11.

Public Health Research Institute, Newark, New Jersey 07103, USA.

The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.
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http://dx.doi.org/10.1074/jbc.M208927200DOI Listing
February 2003