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Clearing tail-anchored proteins from mitochondria  skia, Thomas Beckera,b, and Nikolaus Pfannera,b,1 Łukasz Opalin a Institut für Biochemie und Molekularbiologie and bBIOSS Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany

Eukaryotic cells contain numerous membranebound compartments—the cell organelles. The vast majority of organellar proteins are synthesized as precursors on cytosolic ribosomes and have to be transported to their intracellular destinations (1). The precursor proteins contain organelle-specific targeting signals. Protein machineries located in the cytosol and organelles recognize the precursor proteins and promote their translocation into the organelles. Errors in protein targeting will lead to a mislocalization of proteins with detrimental effects to cell organelles. Thus, in addition to the targeting machineries, quality control systems are important to prevent mistargeting or remove mislocalized proteins from the wrong organelle (2). In PNAS, Okreglak and Walter (3) report the identification of a quality control

system of the mitochondrial outer membrane. Using budding yeast as a model system, they show that a membrane-bound ATPase is a crucial player in a surveillance pathway that detects and extracts mistargeted tailanchored membrane proteins. In the case of the endoplasmic reticulum (ER) and mitochondria, amino-terminal targeting signals are found in a large number of precursor proteins: signal sequences for the signal recognition particle (SRP)-dependent cotranslational translocation into the ER and presequences for posttranslational preprotein translocation into mitochondria (1). However, several further classes of precursor proteins have been identified that contain different types of targeting signals. One class is formed by tail-anchored proteins, which are characterized by a single transmembrane

Fig. 1. Intracellular sorting of tail-anchored proteins. (A) Sorting routes in WT cells. Tail-anchored proteins are synthesized on cytosolic ribosomes and are posttranslationally transported to the targeted organelle. (1) The GET pathway mediates transport and insertion of tail-anchored proteins into the ER membrane. TRC, transmembrane domain-recognition complex. (2) Alternative pathways for insertion of tail-anchored proteins into the ER may operate in particular when the GET pathway is defective. (3) Some mitochondrial (M) tail-anchored proteins use the MIM complex; the biogenesis of other proteins may be facilitated by TOM receptors. (4) An unassisted pathway for insertion into the mitochondrial outer membrane has also been described. (5) The tail-anchored peroxisomal (P) protein Pex15 uses the GET machinery for initial insertion into the ER membrane, followed by vesicular transport to peroxisomes. (6) A direct pathway for peroxisomal tail-anchored proteins involving Pex19 and Pex3 was also described. (B) In cells with a defective GET pathway, Pex15 is mistargeted to the mitochondrial outer membrane and is removed from this compartment with the aid of the AAA-ATPase Msp1 (red arrows). Small amounts of Pex15 can be correctly delivered to peroxisomes (12), possibly by the alternative Pex19/Pex3 pathway. 7888–7889 | PNAS | June 3, 2014 | vol. 111 | no. 22

domain close to the carboxy terminus that functions as a targeting and membrane anchor sequence (4–7). The amino-terminal domain of these proteins is exposed to the cytosol. Tail-anchored proteins comprise ∼5% of integral membrane proteins and are involved in a large variety of functions, including protein translocation, vesicle trafficking, organelle fission, inheritance and motility, and regulation of apoptosis. Tailanchored proteins are targeted to the ER, mitochondria, and peroxisomes in a posttranslational mechanism as the carboxylterminal transmembrane domain leaves the ribosome only on completion of protein synthesis. Tail-anchored proteins thus escape recognition by the cotranslational SRP system and are transported by different transport pathways to their target organelles. The best-studied sorting pathway of tailanchored proteins is the guided entry of tailanchored proteins (GET) pathway into the ER (4, 5, 7, 8). In this pathway, the transmembrane domain of a newly synthesized tail-anchored protein is initially recognized by a cytosolic transmembrane domain recognition complex and transferred to the ATPase Get3 (Fig. 1A). This chaperone cascade shields the hydrophobic transmembrane segment during the journey of the precursor through the cytosol. Get3 delivers the precursor to the ER-resident Get1/Get2 receptor complex that promotes insertion of the precursor into the membrane. In the absence of a functional GET system, additional pathways may be used for insertion of some tail-anchored proteins into the ER [SRP; cytosolic heat shock proteins (Hsp): Hsp40/Hsp70; or unassisted pathway] (4–6). In contrast to the ER, no common import pathway of tail-anchored precursors into the mitochondria has been identified. The translocase of the outer membrane (TOM complex) is the general entry gate for the majority of mitochondrial precursors. However, it is a controversial issue if the TOM complex is Author contributions: Ł.O., T.B., and N.P. wrote the paper. The authors declare no conflict of interest. See companion article on page 8019. 1

To whom correspondence should be addressed. E-mail: Nikolaus. [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1406864111

 ski et al. Opalin

from mitochondria (Fig. 1B). Msp1, an ATPase of the ATPases associated with diverse cellular activities (AAA) family, is located in the mitochondrial outer membrane, as well as in the peroxisomal membrane (3, 17). In an elegant series of experiments, the authors show that Msp1 binds to Pex15 mistargeted to the mitochondria and promotes its removal (3). When the GET pathway is

Okreglak and Walter report the identification of a quality control system of the mitochondrial outer membrane. functional, only small amounts of tailanchored proteins are mislocalized to the mitochondria and are readily removed by Msp1. When the GET pathway is defective, larger amounts accumulate at the mitochondrial outer membrane and disturb mitochondrial functions, including alteration of mitochondrial shape (3, 8); under these conditions, Msp1 is crucial for removing the mistargeted proteins. Double deletion strains of GET components and MSP1 display a strong synthetic growth defect and cause a massive accumulation of Pex15 at mitochondria. Overexpression of Msp1 in the get3 deletion background leads to relocalization of Pex15 into the cytosol, providing strong evidence that Msp1 is required for extraction of the protein from the mitochondrial outer membrane (3). In this quality control function, Msp1 may be related to cell division cycle 48 (Cdc48/p97/

1 Schnell DJ, Hebert DN (2003) Protein translocons: Multifunctional mediators of protein translocation across membranes. Cell 112(4): 491–505. 2 Rodrigo-Brenni MC, Hegde RS (2012) Design principles of protein biosynthesis-coupled quality control. Dev Cell 23(5):896–907. 3 Okreglak V, Walter P (2014) The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins. Proc Natl Acad Sci USA 111:8019–8024. 4 Borgese N, Fasana E (2011) Targeting pathways of C-tail-anchored proteins. Biochim Biophys Acta 1808(3):937–946. 5 Hegde RS, Keenan RJ (2011) Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat Rev Mol Cell Biol 12(12):787–798. 6 Rabu C, Schmid V, Schwappach B, High S (2009) Biogenesis of tailanchored proteins: The beginning for the end? J Cell Sci 122(Pt 20): 3605–3612. 7 Denic V (2012) A portrait of the GET pathway as a surprisingly complicated young man. Trends Biochem Sci 37(10):411–417. 8 Schuldiner M, et al. (2008) The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134(4):634–645. 9 Thornton N, et al. (2010) Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of α-helical outer membrane proteins. J Mol Biol 396(3):540–549. 10 Kemper C, et al. (2008) Integration of tail-anchored proteins into the mitochondrial outer membrane does not require any known import components. J Cell Sci 121(Pt 12):1990–1998. 11 Krumpe K, et al. (2012) Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes. Mol Biol Cell 23(20):3927–3935.

VCP), an AAA-ATPase that removes proteins from the ER membrane and targets them for proteasomal degradation (18). Okreglak and Walter (3) thus identify the first mitochondrial outer membrane protein that is involved in the removal of mislocalized tail-anchored proteins. This mechanism may constitute an important cellular defense system as wrongly localized proteins can hamper mitochondrial function. Future studies will aim at elucidating the molecular mechanisms of substrate recognition and removal by Msp1. How can Msp1 specifically distinguish between mistargeted and authentic mitochondrial tail-anchored proteins? Msp1 is present on the mitochondria and peroxisomes, yet it removes only Pex15 that is mistargeted to the mitochondria and not peroxisomal Pex15. What is the molecular basis for this organelle selectivity of Msp1 and which role is played by peroxisomal Msp1? Thus far, Pex15 is the only identified substrate of the Msp1 quality control pathway. It will be crucial to investigate whether Msp1 exhibits a more common function in the clearance of mislocalized proteins or whether it is specific for Pex15. Because Okreglak and Walter (3) found that a number of mistargeted tailanchored proteins are not degraded via Msp1, the presence of additional pathways that facilitate the degradation of mislocalized tailanchored proteins is likely. Several pathways for ubiquitylation of mitochondrial outer membrane proteins and the recruitment of Cdc48 to the mitochondria have been described (19, 20), which may contribute to the removal of mistargeted tail-anchored proteins from the mitochondria.

12 van der Zand A, Braakman I, Tabak HF (2010) Peroxisomal membrane proteins insert into the endoplasmic reticulum. Mol Biol Cell 21(12): 2057–2065. 13 Lam SK, Yoda N, Schekman R (2010) A vesicle carrier that mediates peroxisome protein traffic from the endoplasmic reticulum. Proc Natl Acad Sci USA 107(50):21523–21528. 14 Hettema EH, Erdmann R, van der Klei I, Veenhuis M (2014) Evolving models for peroxisome biogenesis. Curr Opin Cell Biol 29C:25–30. 15 Horie C, Suzuki H, Sakaguchi M, Mihara K (2002) Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane. Mol Biol Cell 13(5):1615–1625. 16 Beilharz T, Egan B, Silver PA, Hofmann K, Lithgow T (2003) Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae. J Biol Chem 278(10):8219–8223. 17 Nakai M, Endo T, Hase T, Matsubara H (1993) Intramitochondrial protein sorting. Isolation and characterization of the yeast MSP1 gene which belongs to a novel family of putative ATPases. J Biol Chem 268(32): 24262–24269. 18 Brodsky JL (2012) Cleaning up: ER-associated degradation to the rescue. Cell 151(6):1163–1167. 19 Karbowski M, Youle RJ (2011) Regulating mitochondrial outer membrane proteins by ubiquitination and proteasomal degradation. Curr Opin Cell Biol 23(4):476–482. 20 Escobar-Henriques M, Langer T (2014) Dynamic survey of mitochondria by ubiquitin. EMBO Rep 15(3):231–243.

PNAS | June 3, 2014 | vol. 111 | no. 22 | 7889

COMMENTARY

involved in the biogenesis of the tailanchored proteins Bax and Bcl-2 (4). Tailanchored small Tom proteins are inserted into the outer membrane by the mitochondrial import (MIM) complex (9) (Fig. 1A). Moreover, it was reported that integration of some tail-anchored proteins does not require any known mitochondrial protein but depends on the lipid composition of the mitochondrial outer membrane, in particular on a low ergosterol concentration (10, 11). In the latter pathway, tail-anchored proteins may be inserted into the mitochondria by an unassisted default route (independently of proteinaceous transport components). Two import routes of tail-anchored proteins into peroxisomes have been reported (Fig. 1A). The peroxin Pex15 is initially inserted into the ER via the GET pathway and then transferred to peroxisomes via vesicular transport (8, 12, 13). Other tailanchored proteins use an ER-independent pathway, where the precursors are targeted to peroxisomes via the Pex19 cytosolic chaperone and the peroxisome-resident receptor Pex3 (14). The trafficking pathways of tail-anchored precursors are not fully separate, but probably constitute a complex network within the cell. Lack of a functional GET pathway leads to mistargeting of some ER tail-anchored proteins and of Pex15 to mitochondria (3, 8). This mistargeting may be explained in part by related properties of the targeting sequences. The transmembrane domains of mitochondrial tail-anchored proteins are shorter than their ER counterparts and are often flanked by positively charged amino acid residues; however, small alterations in the transmembrane domains are sufficient for mistargeting of the proteins (4, 15, 16). In the cytosol, tail-anchored proteins may thus compete for organelle-specific chaperones/ targeting factors. When the specific targeting machinery is malfunctioning, tail-anchored proteins may use nonnative pathways, leading to their mistargeting to other cellular compartments. Cells are equipped with sophisticated quality control systems that are coupled to biosynthesis factors to allow rapid removal of precursors that fail to properly insert into the target membrane (2). However, a number of precursors can escape this preemptive quality control system and are mislocalized to other organellar membranes. It is unknown how mitochondria deal with mistargeted tail-anchored proteins. Okreglak and Walter (3) shed light on this fundamental question. The authors identify a role of Msp1 (mitochondrial sorting of proteins) in the removal of mistargeted Pex15

Clearing tail-anchored proteins from mitochondria.

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