Summary In response to environmental stress cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. We show further that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical and cellular analyses reveal a new and unexpected function for G proteins as mediators of stress-response signaling.

Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior.

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G 1 phase. Here, we show that the G␣ subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G 2 /M. Accordingly, phosphorylation of Gpa1 in G 2 /M phase leads to polyubiquitination in G 1 phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.

Using differential product entrapment and photolabeling under specifying conditions, we identified a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Cossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring j3-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of &1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring &1,3-glucan synthesis, is coincident with B-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of &1,3-glucan is discussed.

No abstract