Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum

Dyer, John M.; Mullen, Robert T.

doi:10.1016/s0014-5793(01)02315-8

Cited by 84 publications

(66 citation statements)

References 21 publications

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“…This examination revealed that both termini of the desaturase were accessible to cleavage by proteinase K. At the same time, Kar2p, a soluble lumenal resident of the ER, was protected from degradation, indicating that the membrane seal remained intact ( Figure 1D). Taken together, these results indicate that the desaturase is an integral membrane protein of the ER with four transmembrane domains and both N and C termini in the cytosol, which is in agreement with the recently determined topology of a plant orthologue of Ole1p (Dyer and Mullen, 2001).…”

Section: The Desaturase Is An Integral Membrane Protein With Both Tersupporting

confidence: 91%

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Tatzer¹,

Zellnig

Kohlwein

et al. 2002

MBoC

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The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C., and Yaffe, M.P. ). J. Cell Biol. 115, 1249 -1257. We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.

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Section: The Desaturase Is An Integral Membrane Protein With Both Tersupporting

confidence: 91%

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Tatzer¹,

Zellnig

Kohlwein

et al. 2002

MBoC

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“…This morphology was also not observed in any of the cells transformed with myc-FAD2 nor in cells transformed with Arabidopsis FAD2 or Brassica sp. FAD3 (Dyer and Mullen, 2001). These data collectively indicated that both tung FAD2 and FADX were localized exclusively in the ER and that on occasion, transient overexpression of FADX caused a dramatic rearrangement of the ER.…”

Section: Subcellular Localization Of Tung Fad2 and Fadxmentioning

confidence: 58%

“…A model has been proposed in which these membrane-spanning segments function to anchor the enzyme in the endoplasmic reticulum (ER) membrane, with the majority of the protein, including the active site His boxes, exposed on the cytosolic side of the ER ( Fig. 1B; Stukey et al, 1990;Shanklin et al, 1994;Dyer and Mullen, 2001). …”

Section: Sequence Features Of Tung Fad2 and Fadx Proteinsmentioning

confidence: 99%

“…Tobacco (Nicotiana tabacum cv BY-2) suspension culture cells were transiently transformed with DNA encoding either tung FAD2 or FADX, and then cells were processed for indirect immunofluorescence microscopy as described previously (Dyer and Mullen, 2001). In brief, BY-2 cells were biolistically bombarded with plasmid DNA (pRTL2-mycTF2 or pRTL2-mycFADX), and then cells were allowed to recover for 20 to 24 h at 26°C in the dark.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

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Molecular Analysis of a Bifunctional Fatty Acid Conjugase/Desaturase from Tung. Implications for the Evolution of Plant Fatty Acid Diversity

et al. 2002

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The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% ␣-eleostearic acid (18: 3⌬ 9cis,11trans,13trans ), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related ⌬ 12 oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1⌬ 9cis ) into linoleic acid (18:2⌬ 9cis,12cis ) and that FADX converted linoleic acid into ␣-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and ⌬ 12 -desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with ⌬ 12 fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.Conjugated fatty acids are naturally occurring compounds that have specialized uses in nutraceutical and industrial applications. For example, conjugated linoleic acid (CLA) is a potent anticancer compound present in foods derived from ruminant animals (Belury, 2002). This bioactive fatty acid (predominantly the 18:2⌬ 9cis,11trans isomer) is synthesized by rumen bacteria and then absorbed by the animal and concentrated in milk fat or adipose tissue. Rumen bacteria also synthesize 18:1⌬ 11trans , which can be absorbed and then desaturated by an animal stearoyl-CoA desaturase to produce CLA (Corl et al., 2001). Conjugated fatty acids such as ␣-eleostearic acid (18:3⌬ 9cis,11trans,13trans ) have recently shown promise for anticancer applications (Igarashi and Miyazawa, 2000;Kohno et al., 2002), as well as serum lipidlowering effects in mammals (Koba et al., 2002). Oils containing ␣-eleostearic acid may also be used for industrial drying applications. Tung oil, which is derived from seeds of the tung tree (Aleurites fordii Hemsl.), is commonly used in formulations of inks, dyes,...

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“…7,8 In doing so, the Fad3 N-terminal sequences in BF3-GFP-Cb5 and TF3-GFP-Cb5 were orientated towards the cytosol, consistent with their orientation in native (full-length) BF3 and TF3 proteins. 9 A cartoon depicting the structure of the Fad3-GFP-Cb5 fusion proteins, as well as their expected N cytosol -C ER lumen topology is shown in Figure 1A. To measure the degradation rates of the Fad3-GFP-Cb5 fusion proteins in plant cells, we utilized a novel dual fluorescent protein reporter system, as previously described in reference 6.…”

confidence: 99%

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

Khuu

Gidda

Shockey

et al. 2011

Plant Signaling & Behavior

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Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum

Cited by 84 publications

References 21 publications

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Molecular Analysis of a Bifunctional Fatty Acid Conjugase/Desaturase from Tung. Implications for the Evolution of Plant Fatty Acid Diversity

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

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