Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation.
The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Delta12-desaturase) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues of plants. Little is known, however, concerning the post-transcriptional mechanisms that regulate the activity of this important enzyme. The soybean genome possesses two seed-specific isoforms of FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24 amino acid residues. Expression studies in yeast revealed that the FAD2-1A isoform is more unstable than FAD2-1B, particularly when cultures were maintained at elevated growth temperatures. Analysis of chimeric FAD2-1 constructs led to the identification of two domains that appear to be important in mediating the temperature-dependent instability of the FAD2-1A isoform. The enhanced degradation of FAD2-1A at high growth temperatures was partially abrogated by treating the cultures with the 26S proteasome-specific inhibitor MG132, and by expressing the FAD2-1A cDNA in yeast strains devoid of certain ubiquitin-conjugating activities, suggesting a role for ubiquitination and the 26S proteasome in protein turnover. In addition, phosphorylation state-specific antipeptide antibodies demonstrated that the Serine-185 of FAD2-1 sequences is phosphorylated during soybean seed development. Expression studies of phosphopeptide mimic mutations in yeast suggest that phosphorylation may downregulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.
Tang
Guo-Qing
GQ
Crop Science Department, North Carolina State University, Campus Box 7620, Raleigh, NC 27695-7620, USA.
Novitzky
William P
WP
Carol Griffin
H
H
Huber
Steven C
SC
Dewey
Ralph E
RE
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006358:Hot Temperature; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics*
D007527:Isoenzymes; D044943:Fatty Acid Desaturases; C021904:Phosphatidylcholine desaturase; D044925:Oxidoreductases Acting on CH-CH Group Donors