The sunflower plastidial omega3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity.
Although plant plastidial omega3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 omega3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 omega3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.
Venegas-Calerón
Mónica
M
Rothamsted Research, West Common, Harpenden, Herts AL52JQ, UK.
Beaudoin
Frédéric
F
Garcés
Rafael
R
Napier
Johnathan A
JA
Martínez-Force
Enrique
E
D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000378:metabolism*; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D006368:Helianthus / Q000201:enzymology*; D008969:Molecular Sequence Data; D018087:Plastids; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D015525:Fatty Acids, Omega-3; D005288:Ferredoxins; D044943:Fatty Acid Desaturases