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Trends Parasitol. Author manuscript; available in PMC 2017 October 01. Published in final edited form as: Trends Parasitol. 2016 October ; 32(10): 758–760. doi:10.1016/j.pt.2016.05.007.
Toxoplasma retromer is here to stay Olivia L. McGovern and Vern B. Carruthers Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA Vern B. Carruthers: [email protected]
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Abstract How the protozoan pathogen Toxoplasma gondii and related parasites shuttle proteins through their intricate system of endomembranous compartments remains unclear. Sangaré et al. show that the Toxoplasma retromer complex is essential for parasite viability through its role in protein targeting to multiple locales and its interactions with newly identified partners.
Keywords parasite; membrane; protein; trafficking; targeting
Author Manuscript
Similar to other apicomplexan parasites, Toxoplasma gondii possesses an elaborate system of endomembranous compartments in its apical region. These structures include secretory organelles (micronemes, rhoptries and dense granules) of the exocytic system required for invasion, intracellular survival, and egress from host cells. The apical region also contains the endocytic system including endosome-like compartments (ELCs, likely constituting early and late endosomes) and the lysosome-like vacuolar compartment/plant-like vacuole (VAC) [1–3]. Whereas the exo- and endocytic systems are often distinct in other eukaryotes, T. gondiiand potentially other apicomplexans, seem to have merged these systems, at least for the biogenesis of micronemes and rhoptries (Figure 1). The recent finding that T. gondii internalizes exogenous proteins from the host cytoplasm and targets them for degradation in the VAC suggests the parasite also uses its endocytic system for a classic role in protein uptake and degradation [4]. Precisely how proteins are correctly trafficked through the exo/ endocytic system remains poorly understood but is emerging with new insight from Stanislas# Tomavo’s lab interrogating the parasite retromer complex [5].
Author Manuscript
In higher eukaryotes, the retromer complex is a master regulator of intracellular trafficking [6]. The core retromer complex, made up of Vps26, Vps29 and Vps35, associates with other components to form functionally distinct complexes that drive retrograde trafficking of proteins from early and late endosomes to the trans-Golgi network (TGN) or the plasma
Correspondence to: Vern B. Carruthers, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
McGovern and Carruthers
Page 2
Author Manuscript
membrane. Retrograde trafficking effectively recycles cargo receptors and plasma membrane proteins, preventing them from entering the default pathway for degradation in lysosomes. In their new study, the Tomavo group used proteomic and genetic approaches to establish that T. gondii uses a functional retromer complex for retrograde trafficking in its exo/endocytic system [5]. Proteomic experiments identified 17 potential interacting partners of the core retromer complex including TgSortilin (TgSORTLR), Rab5B, Rab11B, and 9 parasitespecific hypothetical proteins, among others. Subsequent genetic knockdown of Vps35 revealed that retromer is essential for parasite viability in culture and in infected mice, and is required for protein trafficking to the secretory organelles and plasma membrane.
Author Manuscript Author Manuscript
Previous work by the Tomavo lab showed that TgSORTLR is a transmembrane endosomal cargo receptor that delivers proteins to the micronemes and rhoptries [1]. Consequently, knockdown of TgSORTLR led to rerouting of microneme and rhoptry proteins to the parasitophorous vacuole (PV), presumably because failure to reach the ELCs redirects these proteins to the constitutive secretion pathway. The current study shows that the retromer complex is required for retrograde trafficking of TgSORTLR from the ELCs to the TGN [5]. Upon disruption of the retromer complex, TgSORTLR cannot return to the TGN to pick up more cargo, leading to mistrafficking of microneme and rhoptry proteins similar to the TgSORTLR knockdown. Unexpectedly, abolishing the retromer complex also perturbed dense granules, suggesting that unlike TgSORTLR, retromer is also required for the dense granule pathway [5]. Additionally, disruption of the retromer complex shifted the predominantly plasma membrane localization of TgHP03, a putative small substrate transporter of the major facilitator superfamily, to vesicular structures within the parasite, implicating the retromer complex in recycling to the plasma membrane [5]. Although the identity of the vesicular structures remains unknown, it will be interesting to determine if TgHP03 and potentially other plasma membrane cargoes are recycled from the ELCs to the plasma membrane. The finding that Rab5B is associated with the parasite retromer complex is also potentially relevant here since its plant ortholog ARA6 promotes endosome to plasma membrane recycling of surface proteins [7].
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The current study also found that Rab11B is associated with the retromer complex, which is intriguing since this small GTPase promotes the biogenesis of the inner membrane complex (IMC), a subpellicular system of endomembranous sacs required for parasite integrity, from the Golgi [8]. Sangaré et al. additionally found that the Golgi or TGN is home to a nonessential retromer partner, TgHP12, which resembles FIP2, a Rab11-interacting protein that promotes trafficking to the plasma membrane in higher eukaryotes [9]. Thus, it will be interesting to determine the extent that interaction of Rab11B and TgHP12 with the retromer complex contributes to formation of the IMC as a possible explanation for loss of parasite integrity upon ablation of the retromer complex. Altogether, the new work suggests the existence of at least four functionally distinct retromer complexes: one that binds TgSORTLR and is required for micronemes and rhoptries, a second that influences the dense granule pathway, a third possibly involving Rab5B for recycling of plasma membrane proteins, and a fourth complex with Rab11B and TgHP12 potentially linked to the IMC (Figure 1).
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
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Author Manuscript
Interestingly, unlike other systems, loss of T. gondii retromer does not lead to lysosomal degradation of retromer-dependent cargo receptors and plasma membrane proteins. Although localization of TgSORTLR and TgHP03 is altered upon disrupting the retromer complex, protein levels were unchanged, suggesting a lack of default targeting to the VAC [5]. Thus, Sangaré et al. conclude that the endosomal system was adapted for biogenesis of the parasite secretory organelles rather than trafficking to and proteolytic degradation within the VAC. However, T. gondii is known to degrade proteins from the host cytosol within the VAC, indicating a degradative role for the endolysosomal system [4]. Precisely how traffic is directed for degradation awaits further investigation, but it is possible that trafficking to the VAC does not occur by default and instead involves receptor-mediated forward targeting, akin to sorting of proteins to the micronemes and rhoptries (Figure 1).
Author Manuscript
The discovery of a functional retromer complex is a substantial advance toward understanding intracellular trafficking in T. gondii. Future work should provide more insight into how distinct retromer complexes direct traffic to the appropriate destination from multiple sites in the exo/endocytic system. The extent that retromer complexes contribute to endocytosis of host-derived proteins also deserves attention. The essential nature of these complexes should spur efforts to understand the functions of additional parasite-specific, retromer-associated hypothetical proteins that can be potential new targets to disrupt trafficking and compromise parasite viability.
References
Author Manuscript Author Manuscript
1. Sloves PJ, et al. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Cell Host Microbe. 2012; 11:515–527. [PubMed: 22607804] 2. Parussini F, et al. Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol. Microbiol. 2010; 76:1340–1357. [PubMed: 20444089] 3. Miranda K, et al. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 2010; 76:1358–1375. [PubMed: 20398214] 4. Dou Z, et al. Toxoplasma gondii ingests and digests host cytosolic proteins. mBio. 2014; 5(4):e01188–e01114. [PubMed: 25028423] 5. Sangare LO, et al. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. Nat. Commun. 2016; 7:11191. [PubMed: 27064065] 6. Burd C, Cullen PJ. Retromer: a master conductor of endosome sorting. Cold Spring Harb. Perspect. Biol. 2014; 6 7. Ebine K, et al. Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants. Small GTPases. 2012; 3:23–27. [PubMed: 22710734] 8. Agop-Nersesian C, et al. Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B. PLoS Pathog. 2010; 6:e1001029. [PubMed: 20686666] 9. Horgan CP, McCaffrey MW. The dynamic Rab11-FIPs. Biochem. Soc. Trans. 2009; 37:1032–1036. [PubMed: 19754446]
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
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Figure 1. Working model for protein targeting within the exo/endocytic system of Toxoplasma
(1) The endosomal sorting receptor TgSORTLR guides microneme and rhoptry proteins through the endosome-like compartments (ELCs), where proteolytic processing may occur before delivery to the microneme (MIC) and rhoptry (ROP) organelles. Subsequent rounds of microneme and rhoptry biogenesis are sustained by retromer-mediated recycling of TgSORTLR from the ELCs to the trans-Golgi network. (2) Retromer also regulates the secretion of dense granules (DGs), although it is unclear if this effect is direct. (3) TgHP03 and potentially other plasma membrane proteins are recycled from internal vesicular
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
Page 5
Author Manuscript
structures (here presumed to involve ELCs) in a retromer-dependent manner. Rab5B may drive this process similar to its plant ortholog ARA6. (4) Retromer interaction with Rab11B and TgHP12, a potential partner of Rab11B, suggests a role for retromer in inner membrane complex (IMC) biogenesis. (5) Depletion of retromer does not result in apparent degradation of retromer cargoes, suggesting that vacuolar compartment/plant-like vacuole (VAC) targeting is not the default pathway in the Toxoplasma endocytic system. If ingested host material traverses the ELCs on its way to the VAC, this may occur in a receptor-mediated manner similar to microneme and rhoptry sorting.
Author Manuscript Author Manuscript Author Manuscript Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
Trends Parasitol. Author manuscript; available in PMC 2017 October 01. Published in final edited form as: Trends Parasitol. 2016 October ; 32(10): 758–760. doi:10.1016/j.pt.2016.05.007.
Toxoplasma retromer is here to stay Olivia L. McGovern and Vern B. Carruthers Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA Vern B. Carruthers: [email protected]
Author Manuscript
Abstract How the protozoan pathogen Toxoplasma gondii and related parasites shuttle proteins through their intricate system of endomembranous compartments remains unclear. Sangaré et al. show that the Toxoplasma retromer complex is essential for parasite viability through its role in protein targeting to multiple locales and its interactions with newly identified partners.
Keywords parasite; membrane; protein; trafficking; targeting
Author Manuscript
Similar to other apicomplexan parasites, Toxoplasma gondii possesses an elaborate system of endomembranous compartments in its apical region. These structures include secretory organelles (micronemes, rhoptries and dense granules) of the exocytic system required for invasion, intracellular survival, and egress from host cells. The apical region also contains the endocytic system including endosome-like compartments (ELCs, likely constituting early and late endosomes) and the lysosome-like vacuolar compartment/plant-like vacuole (VAC) [1–3]. Whereas the exo- and endocytic systems are often distinct in other eukaryotes, T. gondiiand potentially other apicomplexans, seem to have merged these systems, at least for the biogenesis of micronemes and rhoptries (Figure 1). The recent finding that T. gondii internalizes exogenous proteins from the host cytoplasm and targets them for degradation in the VAC suggests the parasite also uses its endocytic system for a classic role in protein uptake and degradation [4]. Precisely how proteins are correctly trafficked through the exo/ endocytic system remains poorly understood but is emerging with new insight from Stanislas# Tomavo’s lab interrogating the parasite retromer complex [5].
Author Manuscript
In higher eukaryotes, the retromer complex is a master regulator of intracellular trafficking [6]. The core retromer complex, made up of Vps26, Vps29 and Vps35, associates with other components to form functionally distinct complexes that drive retrograde trafficking of proteins from early and late endosomes to the trans-Golgi network (TGN) or the plasma
Correspondence to: Vern B. Carruthers, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
McGovern and Carruthers
Page 2
Author Manuscript
membrane. Retrograde trafficking effectively recycles cargo receptors and plasma membrane proteins, preventing them from entering the default pathway for degradation in lysosomes. In their new study, the Tomavo group used proteomic and genetic approaches to establish that T. gondii uses a functional retromer complex for retrograde trafficking in its exo/endocytic system [5]. Proteomic experiments identified 17 potential interacting partners of the core retromer complex including TgSortilin (TgSORTLR), Rab5B, Rab11B, and 9 parasitespecific hypothetical proteins, among others. Subsequent genetic knockdown of Vps35 revealed that retromer is essential for parasite viability in culture and in infected mice, and is required for protein trafficking to the secretory organelles and plasma membrane.
Author Manuscript Author Manuscript
Previous work by the Tomavo lab showed that TgSORTLR is a transmembrane endosomal cargo receptor that delivers proteins to the micronemes and rhoptries [1]. Consequently, knockdown of TgSORTLR led to rerouting of microneme and rhoptry proteins to the parasitophorous vacuole (PV), presumably because failure to reach the ELCs redirects these proteins to the constitutive secretion pathway. The current study shows that the retromer complex is required for retrograde trafficking of TgSORTLR from the ELCs to the TGN [5]. Upon disruption of the retromer complex, TgSORTLR cannot return to the TGN to pick up more cargo, leading to mistrafficking of microneme and rhoptry proteins similar to the TgSORTLR knockdown. Unexpectedly, abolishing the retromer complex also perturbed dense granules, suggesting that unlike TgSORTLR, retromer is also required for the dense granule pathway [5]. Additionally, disruption of the retromer complex shifted the predominantly plasma membrane localization of TgHP03, a putative small substrate transporter of the major facilitator superfamily, to vesicular structures within the parasite, implicating the retromer complex in recycling to the plasma membrane [5]. Although the identity of the vesicular structures remains unknown, it will be interesting to determine if TgHP03 and potentially other plasma membrane cargoes are recycled from the ELCs to the plasma membrane. The finding that Rab5B is associated with the parasite retromer complex is also potentially relevant here since its plant ortholog ARA6 promotes endosome to plasma membrane recycling of surface proteins [7].
Author Manuscript
The current study also found that Rab11B is associated with the retromer complex, which is intriguing since this small GTPase promotes the biogenesis of the inner membrane complex (IMC), a subpellicular system of endomembranous sacs required for parasite integrity, from the Golgi [8]. Sangaré et al. additionally found that the Golgi or TGN is home to a nonessential retromer partner, TgHP12, which resembles FIP2, a Rab11-interacting protein that promotes trafficking to the plasma membrane in higher eukaryotes [9]. Thus, it will be interesting to determine the extent that interaction of Rab11B and TgHP12 with the retromer complex contributes to formation of the IMC as a possible explanation for loss of parasite integrity upon ablation of the retromer complex. Altogether, the new work suggests the existence of at least four functionally distinct retromer complexes: one that binds TgSORTLR and is required for micronemes and rhoptries, a second that influences the dense granule pathway, a third possibly involving Rab5B for recycling of plasma membrane proteins, and a fourth complex with Rab11B and TgHP12 potentially linked to the IMC (Figure 1).
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
Page 3
Author Manuscript
Interestingly, unlike other systems, loss of T. gondii retromer does not lead to lysosomal degradation of retromer-dependent cargo receptors and plasma membrane proteins. Although localization of TgSORTLR and TgHP03 is altered upon disrupting the retromer complex, protein levels were unchanged, suggesting a lack of default targeting to the VAC [5]. Thus, Sangaré et al. conclude that the endosomal system was adapted for biogenesis of the parasite secretory organelles rather than trafficking to and proteolytic degradation within the VAC. However, T. gondii is known to degrade proteins from the host cytosol within the VAC, indicating a degradative role for the endolysosomal system [4]. Precisely how traffic is directed for degradation awaits further investigation, but it is possible that trafficking to the VAC does not occur by default and instead involves receptor-mediated forward targeting, akin to sorting of proteins to the micronemes and rhoptries (Figure 1).
Author Manuscript
The discovery of a functional retromer complex is a substantial advance toward understanding intracellular trafficking in T. gondii. Future work should provide more insight into how distinct retromer complexes direct traffic to the appropriate destination from multiple sites in the exo/endocytic system. The extent that retromer complexes contribute to endocytosis of host-derived proteins also deserves attention. The essential nature of these complexes should spur efforts to understand the functions of additional parasite-specific, retromer-associated hypothetical proteins that can be potential new targets to disrupt trafficking and compromise parasite viability.
References
Author Manuscript Author Manuscript
1. Sloves PJ, et al. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Cell Host Microbe. 2012; 11:515–527. [PubMed: 22607804] 2. Parussini F, et al. Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii. Mol. Microbiol. 2010; 76:1340–1357. [PubMed: 20444089] 3. Miranda K, et al. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 2010; 76:1358–1375. [PubMed: 20398214] 4. Dou Z, et al. Toxoplasma gondii ingests and digests host cytosolic proteins. mBio. 2014; 5(4):e01188–e01114. [PubMed: 25028423] 5. Sangare LO, et al. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. Nat. Commun. 2016; 7:11191. [PubMed: 27064065] 6. Burd C, Cullen PJ. Retromer: a master conductor of endosome sorting. Cold Spring Harb. Perspect. Biol. 2014; 6 7. Ebine K, et al. Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants. Small GTPases. 2012; 3:23–27. [PubMed: 22710734] 8. Agop-Nersesian C, et al. Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B. PLoS Pathog. 2010; 6:e1001029. [PubMed: 20686666] 9. Horgan CP, McCaffrey MW. The dynamic Rab11-FIPs. Biochem. Soc. Trans. 2009; 37:1032–1036. [PubMed: 19754446]
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
Page 4
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Figure 1. Working model for protein targeting within the exo/endocytic system of Toxoplasma
(1) The endosomal sorting receptor TgSORTLR guides microneme and rhoptry proteins through the endosome-like compartments (ELCs), where proteolytic processing may occur before delivery to the microneme (MIC) and rhoptry (ROP) organelles. Subsequent rounds of microneme and rhoptry biogenesis are sustained by retromer-mediated recycling of TgSORTLR from the ELCs to the trans-Golgi network. (2) Retromer also regulates the secretion of dense granules (DGs), although it is unclear if this effect is direct. (3) TgHP03 and potentially other plasma membrane proteins are recycled from internal vesicular
Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
McGovern and Carruthers
Page 5
Author Manuscript
structures (here presumed to involve ELCs) in a retromer-dependent manner. Rab5B may drive this process similar to its plant ortholog ARA6. (4) Retromer interaction with Rab11B and TgHP12, a potential partner of Rab11B, suggests a role for retromer in inner membrane complex (IMC) biogenesis. (5) Depletion of retromer does not result in apparent degradation of retromer cargoes, suggesting that vacuolar compartment/plant-like vacuole (VAC) targeting is not the default pathway in the Toxoplasma endocytic system. If ingested host material traverses the ELCs on its way to the VAC, this may occur in a receptor-mediated manner similar to microneme and rhoptry sorting.
Author Manuscript Author Manuscript Author Manuscript Trends Parasitol. Author manuscript; available in PMC 2017 October 01.
