Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched β-carbon–containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways

6-methoxy gossypol and 6,6'-dimethoxy gossypol were isolated from the seeds and root bark of a St. Vincent Sea Island variety of cotton (AZK-267, GRIN# PI 528406). Crude mixtures of gossypol and the methoxy compounds were obtained by extraction of the tissue with acetone and precipitation with acetic acid. After recrystallization, the preparations were treated with 3-amino-1-propanol to form gossypol Schiff's bases, which were separated by preparative reverse phase chromatography. The separated Schiff's bases were then hydrolyzed with acid, extracted into diethyl ether, concentrated, and precipitated with acetic acid. From the above procedure, both methoxy gossypol compounds were obtained as 1:1 molar acetic acid solvates. Each compound was prepared in sufficient amounts to determine its physical properties and begin testing for bioactivity. Light absorbance differed significantly for the di-3-amino-1-propanol derivatives of gossypol and the methoxy gossypol compounds at 254 nm. Relative response factors were developed, which can be used for determining or correcting analytical measurements of these methylated gossypol forms.

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