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 polyunsaturated 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.

The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells

“…cells other than those exhibiting high levels of ectopic [gene] expression and/or containing karmellae) were calculated and plotted as normalized GFP/rFP fluorescence ratios (±ca. standard deviations) according to O'Quinn et al 6 the results shown in (A-c) are representative of at least three independent experiments. ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

“…Addition of either the BF3 or TF3 N-terminal sequence to GFP-Cb5, however, resulted in enhanced protein degradation that was significantly inhibited by inclusion of MG132 (Fig. 1D and middle and right graphs), indicating the Fad3 N termini contain cis-acting signals that lumenal-targeted red fluorescent protein (RFP), 6 which serves as an internal control to normalize fluorescence values that may change due to variation in expression levels, plasmid copy number, etc., following biolistic bombardment. The degradation rates of each pair of co-expressed proteins (e.g., GFP-Cb5 and RFP-ER) were then calculated by measuring (via microscopy) the GFP/RFP fluorescence ratio in multiple, individually-transformed cells following inhibition of protein translation with cycloheximide.…”

“…6 With the development of the Fad3-GFP-Cb5 reporter constructs described here, we now have in hand a robust set of tools to begin to investigate the possible conserved role of ERAD in the regulation and degradation of Fads in plant cells.…”

“…(d) Graphs illustrating the degradation of GFP-cb5 (left graph), BF3-GFP-cb5 (middle graph) and tF3-GFP-cb5 (right graph) in BY-2 cells. Based on the procedures described in O'Quinn et al 6 cells were biolistically bombarded with dual gene expression binary vectors [as above in (A)] encoding either GFP-cb5, BF3-GFP-cb5, tF3-GFP-cb5 and rFP-er, which served as an internal (fluorescence) control. Following bombardment, cells were incubated for ~3 h to allow for protein expression and intracellular (er) targeting and then cycloheximide (100 μm) was added (at t = 0 h) to block new protein synthesis, along with (or without) the proteasome inhibitor MG132 (100 μm).…”

“…4 The Fad3 proteins are typically short-lived, and their steady-state amount is modulated by temperature through both changes in mRNA translational efficiency as well as alterations in protein half-life. 5,6 Recently, we demonstrated that the half-life of Fad3 proteins is regulated by a combination of cis-acting degradation signals present in their N-terminal regions and proteasomal degradation, 6 but it was not clear whether the N-terminal sequences alone were sufficient for these functions. Here we extend these studies by showing that the N-terminal sequences of two different Fad3 proteins are indeed sufficient to confer rapid, proteasomal-dependent regulation to an ER-localized reporter protein.…”