Abstract
ABSTRACTTail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.
Highlights
Tail-anchored (TA) proteins possess a single transmembrane domain (TMD) close to their C-terminus, which anchors them to cellular membranes and exposes their N-terminal domain to the cytosol
Owing to the few peroxisomal TA proteins identified to date, these studies are based on mammalian PEX26 (PEX15p in yeast) (Buentzel et al, 2015; Halbach et al, 2006; Yagita et al, 2013) and FIS1, which is present on both peroxisomes and mitochondria (Delille and Schrader, 2008; Koch et al, 2005)
We characterize the physicochemical parameters of a large number of TA proteins in mammals and increase the number of bona fide peroxisomal TA proteins significantly, allowing us to identify targeting information and bioinformatically predict targeting
Summary
Tail-anchored (TA) proteins possess a single transmembrane domain (TMD) close to their C-terminus, which anchors them to cellular membranes and exposes their N-terminal domain to the cytosol. Alterations in tail charge and TMD hydrophobicity distribute TA proteins between peroxisomes, mitochondria and ER To verify the bioinformatics results, we first analyzed a selection of ACBD5 mutants (Fig. 3A). With the GFP– ACBD5TMD-T fusions interaction was observed for the wild type (WT), but not for mutants 1–3 suggesting a requirement of high charge and moderate TMD hydrophobicity for PEX19 binding (Fig. 5B). This was confirmed in vitro by testing binding of fluorescently labeled peptides matching the TMD and tail region of ABCD5 to recombinant PEX19 by using fluorescence anisotropy (Fig. 5C). Predicted targeting to the ER was confirmed by expression of Myc–PPP1R3F in COS-7 cells (Fig. 6B)
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