Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

Abstract

Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell's intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane

“…Finally, we comment on the estimated phosphate concentration (160–320 mM; see Table S3 ), which is obtained from fitting of the pH kinetics in vivo. Although this value is larger than the estimated 41 mM intrinsic buffering capacity of a yeast cytosol extract ( 30 ), our value is in good agreement with the total phosphorus concentration of ∼300 mM in yeast (free + bound phosphate groups, organic phosphates, polyphosphate) ( 27 ). We assume that phosphate present in metabolic intermediates (e.g., sugar phosphates) and bound to proteins contributes to the overall buffering, similar to inorganic phosphate; the pKa value of phosphate in sugar phosphates is similar to that of inorganic phosphate.…”

“…The relevant pKa value of CO 2 (aq) is 6.73. We set the pKa of KPi to the pKa of the yeast cytosol as measured in ( 30 ). The external solution, at pH 6, contains 100 mM KPi and 11.3 μ M of CO 2 (aq).…”

Using isoelectric point to determine the pH for initial protein crystallization trials

“…It has been shown that the buffering capacity of the protein itself is negligible both in vitro ( Kirkwood et al. , 2014 ) and in vivo ( Poznanski et al. , 2013 ).…”

Proton Transport and pH Control in Fungi

“…Exchangers can exploit the energy stored in pH gradients to transport ions and solutes against a gradient or to assist in pH control using the gradient of another ion, and may represent the source of the “proton leak” that helps to determine final pH in various organelles [10]. Buffers, particularly the robust phosphate buffering system in S. cerevisiae [11], protect cells and organelles from short-term pH transients, but cannot withstand long-term shifts without assistance from proton transporters [9]. …”