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

Abstract

The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)-localized omega-3 desaturases (Fad3s) are regulated extensively at the post-transcriptional level by both temperature-dependent changes in translational efficiency, as well as modulation of protein half-life. While the N-terminal sequences of Fad3 proteins were shown to contain information that mediates their rapid, proteasome-dependent protein turnover in both plant and yeast cells, it is currently unknown whether these sequences alone are sufficient to direct protein degradation. In this report, we fused the N-terminal sequences of two different Fad3 proteins to an ER-localized fluorescent protein reporter, consisting of the green fluorescent protein and the ER integral membrane protein cytochrome b5, and then measured (via microscopy) the degradation of the resulting fusion proteins in plant suspension-cultured cells relative to a second, co-expressed fluorescent reporter protein. Overall, the results demonstrate that the N-termini of both Fad3 proteins are sufficient for conferring rapid, proteasome-dependent degradation to an ER-bound marker protein.