First published online October 14, 2004; 10.1105/tpc.104.026039
The Plant Cell 16:3002-3019 (2004)
© 2004 American Society of Plant Biologists
Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria
Yeen Ting Hwanga,
Scott M. Pelitireb,
Matthew P.A. Hendersonc,
David W. Andrewsc,
John M. Dyerb,1 and
Robert T. Mullena,1,2
a Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada
b U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana 70124
c Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
2 To whom correspondence should be addressed. E-mail rtmullen{at}uoguelph.ca; fax 519-767-1991.
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 ß-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.
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