Membrane‐bound fatty acid desaturases are inserted co‐translationally into the ER and contain different ER retrieval motifs at their carboxy termini

Abstract

SummaryFatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER). To investigate the biogenesis of ERlocalized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are ef®ciently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and suf®cient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-speci®c motif consisting of F-X-X-K/R/D/E-F-COOH, where -F-are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and de®ne a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.

Two FAD3 Desaturase Genes Control the Level of Linolenic Acid in Flax Seed

“…However, the C-terminal regions of ER-localized desaturases contain ER retrieval motifs (McCartney et al, 2004). Deletion of just five amino acids from the C terminus of the Brassica FAD3 resulted in a dramatic reduction in activity, as well as mislocalization of this protein to the Golgi (McCartney et al, 2004).…”

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…Although these functional complementation results for Cb5 isoforms A, B, and C were expected based upon their localization in the ER in plant cells (Figure 2), the complementation of FAD2 activity by the mitochondrial isoform Cb5-D was surprising. Plant FAD enzymes are localized exclusively in the ER in both plant and yeast cells (Dyer and Mullen, 2001;McCartney et al, 2004), and the ability of Cb5-D to stimulate FAD2 activity suggested that either a proportion of Cb5-D (mis)localized to the ER in yeast cells or that this isoform could donate electrons to ER-bound FADs in trans from the mitochondrial surface. Immunofluorescence microscopic analysis of yeast cells revealed, however, that all four tung myc-tagged Cb5 isoforms localized exclusively to the ER (our unpublished data), indicating that differences in either yeast mitochondrial TA import machinery and/or the cis-acting targeting signals of Cb5-D prevented its sorting to mitochondria.…”

“…One equivalent of microsomes (defined as 100 fmol of SRP receptor a-subunit; Andrews et al, 1989) or one equivalent of mitochondria (defined as 50 mg of total mitochondrial protein) were then added to translation reactions and incubated for 1 h at 248C. Import reactions with microsomes were layered onto a 0.5 M sucrose cushion, and membranes were pelleted by centrifugation at 100,000g for 10 min (McCartney et al, 2004). Gradients were fractionated into top and middle fractions (representing soluble proteins), and a bottom fraction was obtained by solubilizing pelleted material (including microsomes and microsomalassociated proteins) in SDS loading buffer.…”

“…The vectors pSPUTK (Falcone and Andrews, 1991) and pSPUTK-BglII (McCartney et al, 2004) containing the SP6 promoter were used for in vitro membrane insertion experiments. Plasmid pSPUTK/myc-Cb5-A was generated by ligating the NcoI-XbaI fragment from pRTL2/mycCb5-A into NcoI-XbaI-digested pSPUTK.…”

“…Tung is a subtropical tree that produces a seed oil containing high amounts of eleostearic acid (18:3D 9cis,11trans,13trans ), an unusual conjugated fatty acid that imparts industrially useful drying qualities to the oil (Sonntag, 1979). We previously identified the enzyme required for eleostearic acid biosynthesis, a diverged, Cb5-dependent D 12 oleic acid desaturase (FAD2) termed FADX (Dyer et al, 2002), and subsequently determined the mechanisms for FAD targeting and assembly into ER membranes in plant cells (McCartney et al, 2004). Here, we report the identification, functional analysis, and a detailed description of the molecular targeting signals for four Cb5 proteins that are expressed in developing tung seeds during biosynthesis of tung oil.…”