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Intracellular pH distribution and transmembrane pH profile of yeast cells
Abstract:
The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.
Insights
Yeast cells exhibit complex internal pH gradients, with interiors generally alkaline (pH 7.0-7.5). External conditions significantly influence these pH patterns, challenging assumptions in proton transport studies.
Area of Science:
- Microbiology
- Cell Biology
- Biophysics
Background:
- Intracellular pH is a critical parameter influencing cellular functions.
- Previous studies often assumed uniform intracellular pH or relied on bulk measurements.
- Understanding localized pH gradients is essential for studying proton transport and cellular energetics.
Purpose of the Study:
- To investigate the spatial distribution of pH within yeast cells (Saccharomyces cerevisiae and Endomyces magnusii) at high resolution.
- To determine how external pH conditions and cellular metabolic states affect intracellular pH gradients.
- To re-evaluate the validity of using bulk pH measurements for calculating proton electrochemical potential across the plasma membrane.
Main Methods:
- Utilized pH-dependent fluorescence of intracellular fluorescein.
- Achieved a linear resolution of 0.2 microns to map local pH values.
- Examined yeast cells under various conditions: different buffer concentrations and pH, starvation, antimycin treatment, and glucose addition with diethylstilbestrol.
Main Results:
- Yeast cells displayed alkaline interiors (pH 7.0-7.5) with pH decreasing towards the periphery and cell wall in dilute acidic buffers.
- In alkaline buffers, cells showed an alkaline center, acidic ring, and alkaline periphery near the membrane.
- Intracellular pH heterogeneity decreased in starved or antimycin-treated cells.
- Strong buffers led to homogeneous pH patterns (5.5-7.5) within 15-20 minutes.
- Glucose addition did not significantly alter intracellular pH for 20-30 minutes, except with diethylstilbestrol, which increased acidity.
Conclusions:
- The pH difference across the yeast plasma membrane is likely smaller than previously estimated.
- Bulk pH measurements may not accurately reflect the electrochemical potential of protons in cellular transport.
- Localized pH measurements reveal complex intracellular pH heterogeneity crucial for understanding yeast physiology.