July 01, 2022
Yeast are keenly sensitive to internal pH. Several membrane proteins pump H+ ions out of the cell to keep the internal pH near neutral. When carbon becomes scarce, however, it is essential for survival that these pumps get inactivated so the internal space is rapidly acidified. This acidification is postulated to conserve energy and trigger a number of subsequent pathways to combat starvation. Key among these adaptive responses is the derepression of the glucose-repressed genes. The well-studied SWI/SNF complex has been established as a key mediator for this, but the details of how the transcriptional boost is effected have not been known.
In studying the sequence and structure of the eleven subunits of the SWI/SNF complex, the authors noted that ten of the eleven subunits had large intrinsically disordered regions and that four of the eleven contained glutamine-rich low-complexity sequences (QLCs) that contain multiple histidine residues. QLCs were previously identified as important for binding transcription factors.
In looking for a link between pH and activation, the authors postulated that the histidine residues might be important because the histidine sidechain has an intrinsic pKa of 6.9, and thus might change conformation when pH drops.
Detailed comparative analysis of QLCs from yeast and other organisms led the authors to conclude that, in yeast, the histidines are salient features of QLCs that have been evolutionarily conserved. Given this, they noted that the N terminus of Snf5p has one of the largest QLCs in the whole yeast proteome and is in the top three for number of histidines.
Naturally, given the tools of the yeast model, the next step was to mutate the protein, for which they compared a full deletion against an N-terminal deletion of the QLC and a targeted allele with four histidines within the QLC mutated to alanine.
They found that total loss of the gene was phenotypically distinct from either of the QLC-targeted mutants. Total absence of the protein caused disruption of the SWI/SNF architecture, while QLC-mutants maintained an intact complex but showed disruptions in transcriptional reprogramming in response to starvation, as specifically measured by derepression of the ADH2 gene.
By a subsequent series of elegant biochemical experiments—conducted both in vivo and in vitro—the authors show with great precision how the Snf5p QLC specifically senses pH to trigger widespread reprogramming of genes that will help yeast metabolize non-preferred carbon sources. Even more specifically, they show how acidification leads to protonation of the histidines in the QLC, causing that region of the protein to expand and change conformation, thereby affecting the binding properties of the whole SWI/SNF complex.
The ability to do these experiments and develop a model of how the cell accomplishes delicate regulation once more astounds us with the awesome power of yeast genetics.
Categories: Research Spotlight