Abstract
In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24–41 and 508–525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.
Highlights
Alcohol acetyltransferase (AATase; E.C. 2.3.1.84) produces acetate esters through the condensation of an alcohol and acetylcoenzyme A (CoA)
Fluorescent microscopy images of cells overexpressing Atf1-GFP and Erg6-DsRed cultured in oleic acid medium revealed co-localization of GFP and DsRed fluorescence, indicating that Atf1 localized to lipid droplets (LDs) (Figure 1B)
Corresponding to the results from the oleic acid cultures, the N- and C-terminal deletions resulted in cytosolic localization (0% LD localization, i.e. localization was not detected for D2–48, D503–525, and GFP control), confirming the importance of these two regions in membrane association
Summary
Alcohol acetyltransferase (AATase; E.C. 2.3.1.84) produces acetate esters through the condensation of an alcohol and acetylcoenzyme A (CoA). During fermentations 2-phenyl ethanol and CoA condense to phenyl ethyl acetate to produce a flowery aroma reminiscent of roses. Synthesized ethyl and isoamyl acetates produce scents of sweet pear and banana, respectively. In Saccharomyces cerevisiae these reactions are catalyzed by two AATases, Atf and Atf2 [5,6]. Double knockouts of these enzymes eliminated isoamyl ester synthesis and reduced ethyl acetate synthesis by 50% [7]. It has been shown that the transcription of ATF1 is negatively regulated by oxygen and unsaturated fatty acids [10,11] and that fermentations conditions (temperature, nitrogen content and glucose concentration) can significantly alter Atf activity and the resulting volatile ester profiles [12,13]
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