|O'Quin, Jami -|
|Bourassa, Linda -|
|Zhang, Daiyuan -|
|Gidda, Satinder -|
|Chapman, Kent -|
|Mullen, Robert -|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 7, 2010
Publication Date: July 9, 2010
Citation: O'Quin, J.B., Bourassa, L., Zhang, D., Shockey, J.M., Gidda, S.K., Fosnot, S., Chapman, K.D., Mullen, R.T., Dyer, J.M. 2010. Temperature-sensitive, Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway. Journal of Biological Chemistry. 285(28):21781-21796. Interpretive Summary: One of the keys to understanding how to design effective and logical strategies for production of new products in transgenic plants, yeasts, or bacteria rests upon understanding the physical properties of the enzymes to be produced in these hosts. One of the factors that can have a profound affect is growth temperature. In the current study, transgenic yeasts carrying fatty acid desaturase 3 (FAD3) genes from tung tree and canola were grown at different temperatures. Analysis of the amount of FAD3 protein that could be produced and stably maintained revealed major differences between the tung and canola genes; the canola FAD3 was much less stable at 30 degrees Celsius than it was at 20 degrees Celsius, while tung FAD3 was equally stable at both temperatures. In some cases, the sequence of the enzyme in question can determine sensitivity or stability to temperature. The two FAD3 protein sequences were analyzed and a key residue located near the front end of the FAD3 proteins was found that differed between tung FAD3 and many other plant FAD3s, including that of canola. The role of this residue in determining protein stability, as well as the roles of a number of other factors within yeast cells that are also known to regulate protein stability, was studied in detail.
Technical Abstract: Changes in ambient temperature represent a major physiological challenge to poikilothermic organisms that requires rapid adjustments in the composition of cellular membranes in order to preserve overall membrane dynamics and integrity. In plants, the endoplasmic reticulum-localized omega-3 fatty acid desaturases (Fad3s) contribute to this process by increasing the production of polyunsaturated fatty acids at cooler temperatures, but the desaturase genes themselves are typically not upregulated during this adaptive response. Here, we expressed two closely related FAD3 genes in yeast cells that found that differences in their steady-state protein amount of protein correlated directly with differences in amount of fatty acid product formation. The differences in protein abundance were also directly related to differences in protein half-life, rather than mRNA levels, indicating that the proteins are differentially regulated at the post-translational level. Domain swapping experiments demonstrated that a degradation signal was present in the N-terminal region of each protein, and mutagenesis experiments indicated that charge density within a PEST-like sequence was essential for regulating protein half-life. Cultivation of yeast cells at cooler temperatures resulted in a dramatic increase in the half-life of each protein, and microarray array analysis and subsequent expression studies conducted in various mutant yeast strains revealed that the Cdc48 adaptor proteins Doa1 and Shp1, and proteasomal activity, were essential components of Fad3 regulation. Collectively, these results demonstrate that the steady-state amount (and thus associated Fad3 enzyme activity) is modulated in response to temperature through a combination of N-terminal cis-acting degradation signals and specific components of the ER-associated degradation pathway.