Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria

• Published in: The Plant cell Vol. 16; no. 11; pp. 3002 - 3019
• Main Authors: Hwang, Y.T, Pelitire, S.M, Henderson, M.P.A, Andrews, D.W, Dyer, J.M, Mullen, R.T
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01.11.2004
• Subjects: Aleurites - genetics; Aleurites - metabolism; Amino Acid Motifs - genetics; Amino Acid Sequence; amino acid sequences; Amino acids; cell membranes; cytochrome b; Cytochromes; Cytochromes b5 - chemistry; Cytochromes b5 - genetics; Cytochromes b5 - isolation & purification; Cytochromes b5 - metabolism; Cytosol; endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Fatty acids; gene expression; Gene Expression Regulation, Plant - physiology; Intracellular Membranes - metabolism; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mitochondria; Mitochondria - metabolism; Molecular Sequence Data; Phylogeny; Plant cells; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Protein isoforms; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Sorting Signals - genetics; Protein Structure, Tertiary; protein transport; Protein Transport - physiology; Proteins; signal peptide; Signal Transduction - genetics; Vernicia fordii; Yeasts
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.104.026039

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 beta-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.