A Short Regulatory Domain Restricts Glycerol Transport through Yeast Fps1p

Abstract

The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fps1p, a member of the major intrinsic protein (MIP) family of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fps1p. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fps1p and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype.

Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives

“…Resistances are induced to Acetic, but not propionic, sorbic or benzoic acids [19] Propionic, sorbic, benzoic acids, but not acetic acid [1,7] Key induced signalling pathway and activity HOG pathway signalling, leading to Hog1p MAP kinase activation [19] No evidence for a signalling pathway involvement; acid anion may act directly on the War1p transcription factor [13] Key action/target of the induced activity needed for the acquisition of resistance Phosphorylation of Fps1p, leading to Fps1p endocytosis [20] PDR12 gene induction, leading to an elevated plasma membrane level and activity of the Pdr12p ABC transporter [23,28] Probable related stress response Hog1p-directed downregulation of metalloid entry through the Fps1p channel [40] War1p activation in media containing leucine, methionine or phenylalanine as sole nitrogen source [9] Normal physiological function of the major target of the response Fps1p is the major glycerol channel of the plasma membrane, important for osmoadaptation [10,22,38] Pdr12p is also involved in export of the potentially toxic aromatic and branched-chain organic acids produced during amino acid catabolism [9] (2), a MAP kinase constitutively bound to, and that directly phosphorylates, Fps1p (3). This phosphorylation provides the signal for Fps1p endocytosis and degradation, eliminating this source of the acetic acid entry to the cell.…”

“…[19] When activated in these acetic acid-challenged cells, Hog1p phosphorylates Fps1p at two 12 amino acid regions located on the cytosolic surface of the membrane but immediately adjacent to the amino-and carboxy-terminal transmembrane domains of this channel, regions that are also implicated in turgor-mediated channel closure. [10,38] This phosphorylation is the signal for Fps1p to become ubiquitinated, then endocytosed to the vacuole. [20] Fps1p also degraded when, in the absence of applied stress, Hog1p is artificially activated by the expression of a hyperactive allele of its MAP kinase kinase activator (Pbs2 DD ).…”

“…cerevisiae Hog1p is activated transiently both in response to hyperosmotic stress [10,22,38] and with acetic acid stress, [19] but only with the latter stress condition does it trigger a destabilization of Fps1p. [20] The Fps1p aquaglyceroporin rapidly adopts a closed channel conformation in cells shifted to high osmolarity, a Hog1p-independent response to altered cell turgor.…”

“…[20] The Fps1p aquaglyceroporin rapidly adopts a closed channel conformation in cells shifted to high osmolarity, a Hog1p-independent response to altered cell turgor. [10,22,38 There are other differences between the responses of S. cerevisiae to osmostress [10,22,38] and acetic acid stress. [19] The Hog1p activated by acetic acid in low-pH cultures does not cause GPD1 gene induction or an accumulation of intracellular glycerol, events that are characteristic of this MAP kinase becoming activated by hyperosmotic stress.…”

Presence of the Fps1p aquaglyceroporin channel is essential for Hog1p activation, but suppresses Slt2(Mpk1)p activation, with acetic acid stress of yeast

“…Activated in response to hyperosmotic stress (Hedfalk et al, 2004;Pettersson et al, 2005;Tamas et al, 2003), arsenite (Thorsen et al, 2006) and acetic acid (Mollapour & Piper, 2006), it induces physiologically very different responses under these diverse conditions of stress. Only with acetic acid, not hyperosmotic or arsenite stress, has the active Hog1p been shown to trigger endocytosis of Fps1p, the major plasma membrane channel for glycerol, arsenite and undissociated acetic acid (Mollapour & Piper, 2007).…”