Abstract
Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins.
Highlights
Tail-anchored (TA) proteins are a unique class of proteins integral to all cellular membranes and share the defining characteristic of a single transmembrane-domain (TMD) at or near their C terminus (Kutay et al, 1999)
Complete details on the construction procedures used for generating plasmids encoding any of the various A. thaliana TA proteins and all modified versions thereof described in this study are available upon request. pRTL2/MycTOM40 encodes the 40 kDa channel-forming subunit of the A. thaliana the outer membrane of mitochondria (TOM) complex fused to an N-terminal Myc epitope tag (Hwang et al, 2008), pRTL2/BCAT3-Cherry encodes the A. thaliana plastidial branched-chain aminotransferase 3 fused to the monomeric Cherry fluorescent protein (Niehaus et al, 2014), and pRTL2/Cherry-PTS1 encodes the Cherry protein appended to the C-terminal type 1 peroxisomal matrix targeting signal from pumpkin hydroxypyruvate reductase (Ching et al, 2012)
We cross-referenced datasets of authentic A. thaliana mitochondrial membrane proteins previously identified in various proteomics screens (Duncan et al, 2011; Klodmann et al, 2011; reviewed in Duncan et al, 2013) with all of the TA proteins predicted for the A. thaliana deduced proteome (Kriechbaumer et al, 2009; Pedrazzini, 2009; Dhanoa et al, 2010)
Summary
Tail-anchored (TA) proteins are a unique class of proteins integral to all cellular membranes and share the defining characteristic of a single transmembrane-domain (TMD) at or near their C terminus (Kutay et al, 1999). As a consequence of this unique structural feature, the TMD of a TA protein emerges from the ribosome only after the termination of translation. The TA proteins are distinct from membrane proteins that can possess a C-terminal TMD, but, in addition, contain another sequence that initiates translocation into the endoplasmic reticulum (ER) via the classical signal recognition particle (SRP)/Sec co-translational pathway (Grudnik et al, 2009). Some notable examples include the SNAREs (Soluble NSF Attachment protein REceptors), which mediate vesicular transport and fusion (Malsam et al, 2008), subunits of the ER (Osborne et al, 2005), mitochondrial, and plastidial outer membrane translocons (Jarvis et al, 1998; Gutensohn et al, 2000; Werhahn et al, 2001; Allen et al, 2002; Beilharz et al, 2003; Macasev et al, 2004), the electron carrier cytochrome b5(Cb5) (D’Arrigo et al, 1993; Kuroda et al, 1998; Borgese et al, 2001; Hwang et al, 2004), FIS1 (Fission 1), which is required for www.frontiersin.org
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