Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2004
Publication Date: June 1, 2004
Citation: Pelitire, S.M., Hwang, Y.T., Mullen, R.T., Dyer, J.M. FUNCTIONAL ANALYSIS, SUBCELLULAR LOCALIZATION, AND GENE EXPRESSION PATTERNS OF FOUR CYTOCHROME B5 ISOFORMS CLONED FROM DEVELOPING TUNG (ALEURITES FORDII HEMSL.) SEEDS. (Abstract). Technical Abstract: Tung (Aleurites fordii Hemsl.) seed oil is an additive used in the paint and ink industry because of its unique drying properties, mainly due to the oil's unusual fatty acid composition. Since market price and availability vary considerably, generating similar drying oils by expressing tung enzymes and co-factors in a yeast system could be an alternative. Therefore, elucidating the interactions involved in lipid biogenesis and fatty acid desaturation reactions is critical to obtaining useful amounts of industrial product. Cytochrome b5 is a small, heme-binding, obligate electron donor co-factor protein involved in these fatty acid desaturation reactions. Four isoforms of cytochrome b5 were cloned from a cDNA library of developing tung seeds. Immunofluorescent localization studies of BY2 plant cells expressing the tung cytochromes showed that three of the isoforms resided in the endoplasmic reticulum (ER), while the other isoform was located in the mitochondria. Similar studies in yeast revealed that all four isoforms target to the ER, illustrating the possibility of different protein targeting mechanisms. Co-expression of each cytochrome b5 isoform and a tung FAD2 desaturase (responsible for linoleic acid production) in yeast was studied to determine tung cytochrome b5/desaturase interaction and activity. Gas chromatographic analysis of each yeast strain co-expressing the two tung proteins revealed an elevated accumulation of non-endogenous linoleic acid. In addition, tissue specific expression patterns of each isoform by Northern analysis showed that none of the isoforms were specific to the seed, which has been previously shown in tobacco cytochrome b5 studies.