Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b₅ to Either Endoplasmic Reticulum or Mitochondria

Abstract

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

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

“…That is, the ER in these cells exhibited a distinct (i.e., the soluble region just prior to the first predicted transmembrane domain) of Brassica napus (BF3) or tung (Vernicia fordii) (TF3) Fad3 to the N terminus of GFP-Cb5, a well-characterized ER membrane-targeted fusion protein consisting of the green fluorescent protein (GFP) linked to the C-terminal (tail)-anchored (N cytosol -C ER lumen ) ER integral membrane protein cytochrome b 5 (Cb5). 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.…”

“…The degradation rates of each pair of co-expressed proteins (e.g., GFP-Cb5 and RFP-ER) were then calculated by measuring (via microscopy) the GFP/RFP fluorescence ratio in multiple, individually-transformed cells following inhibition of protein translation with cycloheximide. Figure 1B and C demonstrate that each of the GFP-Cb5 fusion proteins colocalized exclusively with the RFP-ER marker protein in the ER of representative transiently-transformed BY-2 cells, as expected, 7,8 and adopted the proper topology in ER membranes, whereby their N termini were oriented towards the cytosol (data not shown for BF3-GFP-Cb5 and TF3-GFP-Cb5). Notably, after extended time periods (e.g.…”

“…the yellow color in the corresponding merged images indicates co-localization of the co-expressed proteins at the er. Also shown (bottom row at the right) are representative images of a GFP-cb5 and rFP-er co-transformed cell, wherein overexpression (~20 h postbombardment) of GFP-cb5 has led to the formation of large, globular fluorescent er structures (indicated with arrowheads), which, based on the appearance of similar cb5-induced er structures in other published studies, 7,12,13 were presumed to be karmellae. (c) topology mapping of the GFP-cb5 reporter protein in er membranes.…”

“…These globular structures were presumed to be karmalle, which are stacks of ER cisternae and are often induced by Cb 5 when overexpressed in eukaryotic cells. 7,12,13 While karmalle do not induce the unfolded protein response, 13 any BY-2 cells exhibiting these types of structures were discarded from our analysis of Fad3-GFP-Cb5 protein half-life. Investigation of protein degradation rates for the various fusion constructs revealed that GFP-Cb5 was moderately stable in plant cells, and notably, the degradation rate was not appreciably affected by inclusion of the proteasomal inhibitor MG132 (Fig.…”

“…(A) cartoon model of the Fad3-GFP-cb5 fusion proteins orientated in an n cytosol -c er lumen manner in the er membrane. As shown, the fusion proteins consist of three domains: (1) the c-terminal (tail)-anchored er membrane protein, cb5 (specifically, cb5 isoform A from tung), 7 including its short lumenal-facing c-terminal region, single c-terminal tMd (colored black) and cytosolic-facing n-terminal region; (2) monomeric GFP 15 (colored green); and (3) the n-terminal, cytosolic-facing soluble domain of Brassica (amino acid residues 1-59) or tung (residues 1-65) Fad3 (BF3 or tF3; colored blue and stippled). (B) targeting and subcellular properties of various GFP fusion proteins transiently expressed in tobacco BY-2 suspension-cultured cells.…”