Temperature-sensitive post-translational regulation of plant omega-3 fatty-acid desaturases is mediated by the endoplasmic reticulum-associated degradation pathway

Abstract

Changes in ambient temperature represent a major physiological challenge to membranes of poikilothermic organisms. In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty-acid desaturases (Fad3) increase the production of poly-unsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not up-regulated during this adaptive response. Here, we expressed two closely related plant FAD3 genes in yeast cells and found that their enzymes produced significantly different amounts of omega-3 fatty acids and that these differences correlated to differences in rates of protein turnover. Domain-swapping and mutagenesis experiments revealed that each protein contained a degradation signal in its N terminus and that the charge density of a PEST-like sequence within this region was largely responsible for the differences in rates of protein turnover. The half-life of each Fad3 protein was increased at cooler temperatures, and protein degradation required specific components of the ER-associated degradation pathway including the Cdc48 adaptor proteins Doa1, Shp1, and Ufd2. Expression of the Fad3 proteins in tobacco cells incubated with the proteasomal inhibitor MG132 further confirmed that they were degraded via the proteasomal pathway in plants. Collectively, these findings indicate that Fad3 protein abundance is regulated by a combination of cis-acting degradation signals and the ubiquitin-proteasome pathway and that modulation of Fad3 protein amounts in response to temperature may represent one mechanism of homeoviscous adaptation in plants.