Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Abstract

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1Ap results in loss of apicoplast protein import and subsequent death of the parasite. Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1Ap results in loss of apicoplast protein import and subsequent death of the parasite. IntroductionApicomplexa are a phylum of obligate parasites that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Recent evidence suggests that apicomplexans evolved from a free-living photosynthetic ancestor (1.Moore R.B. Oborník M. Janouskovec J. Chrudimský T. Vancová M. Green D.H. Wright S.W. Davies N.W. Bolch C.J. Heimann K. Slapeta J. Hoegh-Guldberg O. Logsdon J.M. Carter D.A. Nature. 2008; 451: 959-963Crossref PubMed Scopus (343) Google Scholar). This ancestry is reflected in the presence of a chloroplast-like organelle, the apicoplast (2.Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (610) Google Scholar). While no longer engaged in photosynthesis, the apicoplast is essential to parasite survival and home to several critical biosynthetic pathways. Bioinformatic and experimental evidence suggests that the apicoplast is engaged in the synthesis of fatty acids, isoprenoids, and heme (3.Ralph S.A. van Dooren G.G. Waller R.F. Crawford M.J. Fraunholz M.J. Foth B.J. Tonkin C.J. Roos D.S. McFadden G.I. Nat. Rev. Microbiol. 2004; 2: 203-216Crossref PubMed Scopus (507) Google Scholar, 4.Fichera M.E. Roos D.S. Nature. 1997; 390: 407-409Crossref PubMed Scopus (498) Google Scholar). Genetic or pharmacological ablation of these pathways blocks parasite growth and the apicoplast, therefore, is currently considered a prime target for antiparasitic drug development (5.Mazumdar J. Wilson E. Masek K.A. Hunter C. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13192-13197Crossref PubMed Scopus (180) Google Scholar, 6.Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Türbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1021) Google Scholar, 7.Goodman C.D. McFadden G.I. Current Drug Targets. 2007; 8: 15-30Crossref PubMed Scopus (75) Google Scholar). The organelle was derived by secondary endosymbiosis and reflects the successful union of a red alga and a heterotrophic eukaryote (8.Gould S.B. Waller R.F. McFadden G.I. Annu Rev. Plant Biol. 2008; 59: 491-517Crossref PubMed Scopus (484) Google Scholar). A large number of algal genes were transferred to the host nucleus, and their products must now be routed back into the organelle. Trafficking occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence (9.Waller R.F. Keeling P.J. Donald R.G. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12352-12357Crossref PubMed Scopus (635) Google Scholar). Transport from the ER to the apicoplast is believed to be vesicle mediated and to sidestep the Golgi apparatus (10.DeRocher A. Gilbert B. Feagin J.E. Parsons M. J. Cell Sci. 2005; 118: 565-574Crossref PubMed Scopus (57) Google Scholar, 11.Tonkin C.J. Struck N.S. Mullin K.A. Stimmler L.M. McFadden G.I. Mol. Microbiol. 2006; 61: 614-630Crossref PubMed Scopus (77) Google Scholar). Once delivered to the apicoplast, proteins have to cross three additional membranes. Recently, we demonstrated that a homolog of Tic20, a component of the translocon of the inner chloroplast membrane (Tic) 4The abbreviations used are: Tictranslocon of the inner chloroplast membraneERADendoplasmic reticulum-associated protein degradationRACErapid amplification of cDNA endsHAhemagglutininGFPgreen fluorescent proteinACPacyl-carrier proteinPIPES1,4-piperazinediethanesulfonic acidATcanhydrotetracyclineToctranslocon of the outer chloroplast membraneCATchloramphenicol acetyltransferaseRFPred fluorescent protein. complex in plants, is likely required for protein import across the innermost membrane of the apicoplast (12.van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13574-13579Crossref PubMed Scopus (147) Google Scholar). To date bioinformatics searches have failed to identify a matching translocon of the outer chloroplast membrane in apicomplexans (13.Tonkin C.J. Kalanon M. McFadden G.I. Traffic. 2008; 9: 166-175PubMed Google Scholar). Analysis of the genome of the remnant nucleus of the algal endosymbiont in cryptomonads showed the presence of an ER-associated protein degradation (ERAD) system and offered a candidate for a translocon across the third and potentially the second membrane (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar). Classically, ERAD functions in the homeostasis of the secretory pathway by retrieving misfolded proteins from the ER that are subsequently degraded by the cytoplasmic proteasome. Der1 is a candidate for the membrane protein pore of the translocon (15.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar). The ATPase Cdc48 together with its cofactors Ufd1 and Npl4 recognizes substrate emerging on the cytosolic side of the ER (16.Meyer H.H. Shorter J.G. Seemann J. Pappin D. Warren G. EMBO J. 2000; 19: 2181-2192Crossref PubMed Scopus (381) Google Scholar) (Fig. 7a). ATP hydrolysis then drives extraction into the cytoplasm followed by degradation in the proteasome (17.Flierman D. Ye Y. Dai M. Chau V. Rapoport T.A. J. Biol. Chem. 2003; 278: 34774-34782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Recently Der1 homologs have been shown to localize to the plastids of a range of secondary plastid-containing organisms (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar, 19.Spork S. Hiss J.A. Mandel K. Sommer M. Kooij T.W. Chu T. Schneider G. Maier U.G. Przyborski J.M. Eukaryot. Cell. 2009; 8: 1134-1145Crossref PubMed Scopus (114) Google Scholar, 20.Kalanon M. Tonkin C.J. McFadden G.I. Eukaryot. Cell. 2009; 8: 1146-1154Crossref PubMed Scopus (66) Google Scholar), but evidence linking these proteins to a functional role in apicoplast import is lacking. Here we use Toxoplasma as a genetic model to rigorously test the function of an ERAD-derived system in apicoplast biogenesis. We demonstrate the presence of two differentially localized systems, one in the ER/cytoplasm and one in the apicoplast. These systems are phylogenetically distinct and the apicoplast ERAD is derived from the algal endosymbiont. Importantly, we provide direct genetic and biochemical evidence for an essential function of the ERAD membrane component Der1Ap in apicoplast protein import.DISCUSSIONEndosymbiosis is now well established as a mechanism that has played a crucial role in the evolution of eukaryotic cells. One organism, the symbiont, is engulfed by a second organism, the host, and a stable symbiotic relationship ensues in which the endosymbiont loses its independence and gradually evolves into an organelle that is controlled by the host and serves the host metabolic needs. A common hallmark of this process is massive horizontal gene transfer from the endosymbiont to the host (43.Kleine T. Maier U.G. Leister D. Annu Rev. Plant. Biol. 2009; 60: 115-138Crossref PubMed Scopus (234) Google Scholar). This gene transfer affords control to the host but also requires the establishment of mechanisms to reroute proteins that are now encoded and synthesized by the host back into the symbiont. A large body of work on mitochondria and chloroplasts has demonstrated the presence of elaborate protein translocons in the inner and outer membranes of these organelles (44.Neupert W. Herrmann J.M. Annu. Rev. Biochem. 2007; 76: 723-749Crossref PubMed Scopus (1081) Google Scholar, 45.Jarvis P. New Phytol. 2008; 179: 257-285Crossref PubMed Scopus (287) Google Scholar) that specifically recognize targeting information and deliver cargo proteins accordingly to the organellar lumen or various membrane compartments.The apicoplast is the product of secondary endosymbiosis, the enslavement of a single celled eukaryotic alga. Compared with their primary progenitors, secondary plastids are surrounded by additional membranes that must be traversed by nuclear-encoded proteins (a total of four membranes surround the apicoplast). The evolution of mechanisms to traverse these additional membranes must have occurred early in organelle acquisition, and a simple solution would have been to use existing protein transport complexes. Our previous studies support this model and have shown that transport over the innermost apicoplast membrane is dependent on elements derived from the translocon of the inner chloroplast membrane of the endosymbionts chloroplast (12.van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13574-13579Crossref PubMed Scopus (147) Google Scholar). The conservation of the Tic complex would make it appear likely that the Toc complex is equally conserved. However, so far genome searches have failed to identify Toc components in Apicomplexa or diatoms (13.Tonkin C.J. Kalanon M. McFadden G.I. Traffic. 2008; 9: 166-175PubMed Google Scholar). The presence of the Toc complex might be masked by a high level of sequence divergence of its components or alternatively indicate that it has been replaced by a different mechanism. How proteins might cross the third or periplastid membrane is of particular interest as this membrane is thought to be a derivative of the endosymbionts plasma membrane. Based on the discovery of genes encoding elements of the ERAD system in the nucleomorph in cryptophytes, Sommer et al. (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar) proposed that this complex was retooled to function in trafficking of plastid proteins (proteins of ERAD origin have also been speculated to be part of the peroxisomal proteome (46.Gabaldón T. Snel B. van Zimmeren F. Hemrika W. Tabak H. Huynen M.A. Biol. Direct. 2006; 1: 8Crossref PubMed Scopus (142) Google Scholar)). Most recently, this model has received support from studies on a variety of organisms bearing secondary plastids including the current study. ERAD-associated proteins are found in the membranous compartment surrounding secondary plastids in the apicomplexans Plasmodium and Toxoplasma ((19.Spork S. Hiss J.A. Mandel K. Sommer M. Kooij T.W. Chu T. Schneider G. Maier U.G. Przyborski J.M. Eukaryot. Cell. 2009; 8: 1134-1145Crossref PubMed Scopus (114) Google Scholar, 20.Kalanon M. Tonkin C.J. McFadden G.I. Eukaryot. Cell. 2009; 8: 1146-1154Crossref PubMed Scopus (66) Google Scholar) and Figs. 1 and 2 and supplemental Fig. S1), the diatom Phaeodactylum (19.Spork S. Hiss J.A. Mandel K. Sommer M. Kooij T.W. Chu T. Schneider G. Maier U.G. Przyborski J.M. Eukaryot. Cell. 2009; 8: 1134-1145Crossref PubMed Scopus (114) Google Scholar), and the cryptophyte Guillardia (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar). Light and electron microscopy experiments indicate that these proteins are associated with the outer membranes of the plastid, but they lack resolution to tie the system to a specific membrane. Interestingly, split GFP assays in Phaeodactylum demonstrate the presence of two Der1 homologs in the third membrane. However, it is important to note that these experiments do not exclude the presence of Der1 homologs in other plastid membranes. Additional markers are needed to establish if the ERAD system is limited to the third membrane or might also be found in the second membrane and thus have replaced the Toc complex previously present in this membrane (Fig. 7d).Whereas the presence of an ERAD translocon in the membranes of plastids was consistent with a role in plastid biology, it was unclear what this role of the ERAD complex might be. Functional data discriminating between a role in protein import versus protein export and quality control have as yet been missing. Whereas symbiont-derived Der1 proteins in Phaeodactylum have been shown to interact at a steady state level with fusion proteins targeted to the periplastid compartment, no interaction was observed with fusion proteins targeted to the plastid lumen (38.Hempel F. Bullmann L. Lau J. Zauner S. Maier U.G. Mol. Biol. Evol. 2009; 26: 1781-1790Crossref PubMed Scopus (88) Google Scholar). In the current study, we have used the ability to construct conditional mutants in T. gondii to devise a rigorous test of the protein import hypothesis. We isolated a T. gondii Der1Ap mutant and demonstrated that this protein is essential for apicoplast protein import and parasite survival. We have previously shown that apicoplast protein import across the innermost membrane is essential for Toxoplasma survival (12.van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13574-13579Crossref PubMed Scopus (147) Google Scholar). In contrast, genetic disruption of classical ERAD in yeast or mammalian systems does not affect cell viability (18.Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar), and mutant cells have to be subjected to stressors resulting in the accumulation of misfolded proteins to produce a viability phenotype. More directly, we have developed and validated assays that track post-translational modifications of reporters that are restricted to the apicoplast lumen and thus require successful import (12.van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13574-13579Crossref PubMed Scopus (147) Google Scholar). Applying these assays to the current study, we demonstrate a direct correlation between the loss of Der1Ap and a complete loss of apicoplast protein import (Fig. 5). These genetic and biochemical experiments provide the strongest evidence to date that the novel plastid ERAD system has a direct and essential role in plastid protein import.Our phylogenetic analyses of CDC48 and Ufd1 indicate that the T. gondii ERAD systems are phylogenetically distinct and that the apicoplast system is derived from the red algal endosymbiont (Fig. 6 and supplemental Fig. S4). This is consistent with the presence of homologs in the cryptophyte nucleomorph, a remnant of the algal nucleus (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar). It thus appears that the Der1 protein of the red algal symbiont was re-targeted from its original location in the ER to the symbiont to its plasma membrane, where it could now function in importing plastid-targeted proteins from the host secretory pathway (Fig. 7, a–c). This represents a remarkably simple and elegant solution for the complex problem of how to evolve protein exchange between host and endosymbiont at the beginning of their relationship. Once targeted to the endosymbionts cytoplasm proteins could take advantage of established mechanisms, namely the Toc and Tic, to gain access to the chloroplast. We hypothesize that the apicoplast employs a series of specific translocons that reflect the diverse evolutionary origin of the membranes in which they reside (Fig. 7d). IntroductionApicomplexa are a phylum of obligate parasites that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Recent evidence suggests that apicomplexans evolved from a free-living photosynthetic ancestor (1.Moore R.B. Oborník M. Janouskovec J. Chrudimský T. Vancová M. Green D.H. Wright S.W. Davies N.W. Bolch C.J. Heimann K. Slapeta J. Hoegh-Guldberg O. Logsdon J.M. Carter D.A. Nature. 2008; 451: 959-963Crossref PubMed Scopus (343) Google Scholar). This ancestry is reflected in the presence of a chloroplast-like organelle, the apicoplast (2.Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (610) Google Scholar). While no longer engaged in photosynthesis, the apicoplast is essential to parasite survival and home to several critical biosynthetic pathways. Bioinformatic and experimental evidence suggests that the apicoplast is engaged in the synthesis of fatty acids, isoprenoids, and heme (3.Ralph S.A. van Dooren G.G. Waller R.F. Crawford M.J. Fraunholz M.J. Foth B.J. Tonkin C.J. Roos D.S. McFadden G.I. Nat. Rev. Microbiol. 2004; 2: 203-216Crossref PubMed Scopus (507) Google Scholar, 4.Fichera M.E. Roos D.S. Nature. 1997; 390: 407-409Crossref PubMed Scopus (498) Google Scholar). Genetic or pharmacological ablation of these pathways blocks parasite growth and the apicoplast, therefore, is currently considered a prime target for antiparasitic drug development (5.Mazumdar J. Wilson E. Masek K.A. Hunter C. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13192-13197Crossref PubMed Scopus (180) Google Scholar, 6.Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Türbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1021) Google Scholar, 7.Goodman C.D. McFadden G.I. Current Drug Targets. 2007; 8: 15-30Crossref PubMed Scopus (75) Google Scholar). The organelle was derived by secondary endosymbiosis and reflects the successful union of a red alga and a heterotrophic eukaryote (8.Gould S.B. Waller R.F. McFadden G.I. Annu Rev. Plant Biol. 2008; 59: 491-517Crossref PubMed Scopus (484) Google Scholar). A large number of algal genes were transferred to the host nucleus, and their products must now be routed back into the organelle. Trafficking occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence (9.Waller R.F. Keeling P.J. Donald R.G. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12352-12357Crossref PubMed Scopus (635) Google Scholar). Transport from the ER to the apicoplast is believed to be vesicle mediated and to sidestep the Golgi apparatus (10.DeRocher A. Gilbert B. Feagin J.E. Parsons M. J. Cell Sci. 2005; 118: 565-574Crossref PubMed Scopus (57) Google Scholar, 11.Tonkin C.J. Struck N.S. Mullin K.A. Stimmler L.M. McFadden G.I. Mol. Microbiol. 2006; 61: 614-630Crossref PubMed Scopus (77) Google Scholar). Once delivered to the apicoplast, proteins have to cross three additional membranes. Recently, we demonstrated that a homolog of Tic20, a component of the translocon of the inner chloroplast membrane (Tic) 4The abbreviations used are: Tictranslocon of the inner chloroplast membraneERADendoplasmic reticulum-associated protein degradationRACErapid amplification of cDNA endsHAhemagglutininGFPgreen fluorescent proteinACPacyl-carrier proteinPIPES1,4-piperazinediethanesulfonic acidATcanhydrotetracyclineToctranslocon of the outer chloroplast membraneCATchloramphenicol acetyltransferaseRFPred fluorescent protein. complex in plants, is likely required for protein import across the innermost membrane of the apicoplast (12.van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13574-13579Crossref PubMed Scopus (147) Google Scholar). To date bioinformatics searches have failed to identify a matching translocon of the outer chloroplast membrane in apicomplexans (13.Tonkin C.J. Kalanon M. McFadden G.I. Traffic. 2008; 9: 166-175PubMed Google Scholar). Analysis of the genome of the remnant nucleus of the algal endosymbiont in cryptomonads showed the presence of an ER-associated protein degradation (ERAD) system and offered a candidate for a translocon across the third and potentially the second membrane (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar). Classically, ERAD functions in the homeostasis of the secretory pathway by retrieving misfolded proteins from the ER that are subsequently degraded by the cytoplasmic proteasome. Der1 is a candidate for the membrane protein pore of the translocon (15.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar). The ATPase Cdc48 together with its cofactors Ufd1 and Npl4 recognizes substrate emerging on the cytosolic side of the ER (16.Meyer H.H. Shorter J.G. Seemann J. Pappin D. Warren G. EMBO J. 2000; 19: 2181-2192Crossref PubMed Scopus (381) Google Scholar) (Fig. 7a). ATP hydrolysis then drives extraction into the cytoplasm followed by degradation in the proteasome (17.Flierman D. Ye Y. Dai M. Chau V. Rapoport T.A. J. Biol. Chem. 2003; 278: 34774-34782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Recently Der1 homologs have been shown to localize to the plastids of a range of secondary plastid-containing organisms (14.Sommer M.S. Gould S.B. Lehmann P. Gruber A. Przyborski J.M. Maier U.G. Mol. Biol. Evol. 2007; 24: 918-928Crossref PubMed Scopus (121) Google Scholar, 19.Spork S. Hiss J.A. Mandel K. Sommer M. Kooij T.W. Chu T. Schneider G. Maier U.G. Przyborski J.M. Eukaryot. Cell. 2009; 8: 1134-1145Crossref PubMed Scopus (114) Google Scholar, 20.Kalanon M. Tonkin C.J. McFadden G.I. Eukaryot. Cell. 2009; 8: 1146-1154Crossref PubMed Scopus (66) Google Scholar), but evidence linking these proteins to a functional role in apicoplast import is lacking. Here we use Toxoplasma as a genetic model to rigorously test the function of an ERAD-derived system in apicoplast biogenesis. We demonstrate the presence of two differentially localized systems, one in the ER/cytoplasm and one in the apicoplast. These systems are phylogenetically distinct and the apicoplast ERAD is derived from the algal endosymbiont. Importantly, we provide direct genetic and biochemical evidence for an essential function of the ERAD membrane component Der1Ap in apicoplast protein import.