Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

Abstract

Adaptation of cells to acetic acid requires a hitherto unknown number of proteins. Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid. Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family). Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y. lipolytica. Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid. Expression of GPR1 is induced by acetic acid and moderately repressed by glucose. It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions. Fluorescence microscopy confirmed a localization to the plasma membrane. A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.

The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

“…Acetate (pK a ϭ 4.76) is known to cross cell membranes in its protonated state and then release the proton into the cytoplasm (46,47). Therefore, either the cyto-plasm of sta6 cells is at a higher resting pH and/or better buffered than that of cw15 cells, or the sta6 strain is for some reason not responsive to some feature of the deflagellation/reflagellation signal, hypotheses we plan to test.…”

The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome

“…Therefore, we constructed an ato5⌬ mutant in the prototrophic SC5314 strain using the SAT-flipper methodology (31). We have subsequently defined a glucose-free minimal medium, YNB, containing 0.5% allantoin as the nitrogen source and 2% Casamino acids as the sole carbon source (YAC), which supports more robust alkalinization (24,25), and tested both the new ato5⌬ mutant and a point mutant in ATO1 (ATO1 G53D ) that has dominant-negative phenotypes in other systems (33).…”

“…Briefly, ϳ1,000 bp of the ACT1 promoter from pAU34 (32) was subcloned between the KpnI and XhoI sites in CIp10 to generate pHZ116. Then, the ATO1 G53D mutation was generated by sitedirected overlap PCR using complementary oligonucleotides with a single mismatch to encode a change of Gly-53 to Asp, analogous to the Y. lipolytica GPR1-1 mutant originally identified by Augstein et al (33), and cloned into pHZ116 to generate pML341. The plasmid was digested with StuI and used to transform CAI4-F2 to uridine prototrophy.…”

The Gpr1/Fun34/YaaH Protein Family in the Nonconventional Yeast Yarrowia lipolytica and the Conventional Yeast Saccharomyces cerevisiae

“…G248D and L65Q (Gpr1-1 and Gpr1-2, respectively) amino acid substitution mutagenesis leads to a dominant acetic acid sensitivity, which was shown by characterisation of the acetic acid sensitive mutants B204-12C-156 and B204-12C-112 (Augstein 2001). Due to further mutagenesis experiments, mutations of F61L, G62S, G63D, N66S, S197 and T198 were found to exhibit acetic acid hypersensitivity, too (Kuschel 2006).…”

“…It is an intra-membrane protein consisting of five to six putative transmembrane domains. Localisation approaches by GFP fusion under the control of the native Gpr1 promoter showed plasma membrane localisation after 2 h and increasing vesicular and vacuolar localisation, which may be connected with recycling and/or degradation processes of the protein, after 4 h of incubation in 30 mM acetic acid-containing medium (Tzschoppe 1998;Augstein 2001;Gentsch 2005). Furthermore, the mutant forms of Gpr1p are located in the plasma membrane (Tzschoppe 1998).…”