bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The Rab11-Family Interacting Proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

Running Head: Toxoplasma PV-mammalian Rab11 vesicles

Eric J. Hartman, Julia D. Romano*, Isabelle Coppens*

Department of Molecular Microbiology and Immunology Johns Hopkins School of Public Health 615N Wolfe Street Baltimore, MD 21205, USA

*equally senior authors. Correspondence addressed to: [email protected]; [email protected]

Main abbreviations: ABD, Arf6-binding domain; ERC, endocytic recycling compartment; FIP, Rab11- Family Interacting protein; IVN, intravacuolar network; PV, parasitophorous vacuole; RBD, Rab11- binding domain; RE, recycling endosomes; SE, sorting endosomes; TfR, transferrin receptor. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract

After invasion of mammalian cells, the parasite Toxoplasma gondii multiplies in a self-made membrane- bound compartment, the parasitophorous vacuole (PV). We previously showed that intravacuolar Toxoplasma interacts with many host cell organelles, especially recycling endosomes, and further manipulates the host endocytic recycling through the sequestration of Rab11 vesicles into the PV. Mammalian Rab-PV interactions are likely mediated by Toxoplasma and host proteins that remain to be identified. In this context, we have examined the specificity of host Rab vesicle interaction with the PV by monitoring the recruitment of subtypes of Rab11 vesicles differing in their composition in Rab11-Family Interacting Proteins (FIPs). We found that vesicles with FIPs from Class I (FIP1C, FIP2, FIP5) or Class II (FIP3, FIP4) are distributed at the PV and detected to varying degrees inside the PV. The PV delivery of vesicles with FIPs from Class I, but not Class II, is Rab11-dependent. In addition to Rab11, FIP3 binds to Arf6, and vesicles associated with FIP3-Arf6 complexes are observed within the PV. Binding of FIP3 to either Rab11 or Arf6 significantly increases the internalization of vesicles into the PV. These data point to a selective process of host recycling endosome recognition and scavenging mediated by Toxoplasma. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Introduction

The small monomeric Rab GTPases belong to the Ras superfamily and are master regulators of vesicular trafficking and signaling pathways (Chavrier and Goud, 1999). Many intracellular pathogens hijack the host Rab membrane trafficking machinery to thrive in a hostile environment. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a protective, nonfusogenic membrane-bound compartment, the parasitophorous vacuole (PV). Recent evidence demonstrates that apicomplexan parasites manipulate several mammalian Rab vesicle trafficking pathways to scavenge nutrients or to exploit the functions of Rab GTPase proteins for immune evasion (reviewed in Coppens and Romano, 2020). The apicomplexan Toxoplasma gondii, the etiologic agent of toxoplasmosis, diverts many host Rab vesicles to the PV and sequesters some of them in the PV lumen through deep invaginations of the PV membrane (Romano et al., 2013; 2017; Nolan et al., 2017; Coppens and Romano, 2020). Toxoplasma predominantly hijacks the GTP-bound form of host Rab proteins to intercept vesicular traffic as a means for lipid procuration. Indeed, the parasite salvages sphingolipids manufactured in the host ER/Golgi and accesses these lipids through the interception of Rab14, Rab30 and Rab43 vesicles (Romano et al., 2013). The Rab11 proteins, comprising Rab11A, Rab11B and Rab11C/Rab25, localize to the trans-Golgi network (TGN), post-Golgi vesicles and pericentriolar recycling endosomes (RE), and they function in the recycling of material to the plasma membrane via RE (Welz et al., 2014; Grant and Donaldson, 2009). Rab11 proteins regulate the recycling of many receptors and adhesion proteins to the cell surface and play roles in diverse cellular functions, including ciliogenesis, cytokinesis, neuritogenesis and oogenesis (Kelly et al., 2012). Abnormal Rab11 activity (deficiency or overexpression) has been shown to be implicated in the progression of numerous human diseases (e.g., cancers, neurodegeneration, diabetes) (Bhuin and Roy, 2015). Rab11A, Rab11B and Rab11C/Rab25 localize to different cellular compartments, suggesting division of selective functions between the three subfamily members. Rab11A is involved in the targeted delivery of material to the cleavage furrow/midbody during cell division, phagocytosis, long-term potentiation of synaptic transmission, and cell migration (Horgan et al., 2012; Cox et al., 2000; Wang et al., 2008; Kelly et al., 2011; Yoon et al., 2005; Caswell et al., 2008) while Rab11B is essential for the recycling of the transferrin receptor (TfR) (Schlierf et al., 2000), the epithelial sodium channel, the cystic fibrosis transmembrane conductance regulator, for the regulation of the trafficking of vacuolar type H+- ATPases, and for calcium-induced exocytosis (Khvotchev et al., 2003; Silvis et al., 2009; Sugawara et al., 2009; Best et al., 2011; Butterworth et al., 2012). Rab11C/Rab25 is associated with apical RE and regulates the processes of transcytosis and plasma membrane recycling (Casanova et al., 1999). Among mammalian Rab vesicles, those from the recycling and secretory (anterograde) pathways are detected in bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

the vast majority of the Toxoplasma PV (Romano et al., 2017). In particular, 89% and 100% of PV contain Rab11A and Rab11B vesicles, respectively. Rab11 proteins interact with different effectors depending on their GTP/GDP status that is regulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP) (Zerial and McBride, 2001). In the active state, Rab11 proteins bind to effector proteins termed the Rab11-Family Interacting Proteins (FIPs), allowing Rab11 proteins to recruit cellular motor proteins (Horgan and McCaffrey, 2009). FIPs interact indiscriminately with GTP-bound Rab11A and Rab11B via a conserved 20-aa C-terminal domain, named the Rab11-binding domain (RBD) (Junutula et al., 2004; Horgan and McCaffrey, 2009; Eathiraj et al., 2006; Jagoe et al., 2006). Mutations introduced into the RBD of FIP disrupts binding to Rab11, and therefore alters the localization of the FIP (Lindsay and McCaffrey 2004a; Junutula et al., 2004; Jagoe et al., 2006; Wilson et al., 2005; Fielding et al., 2005; Horgan et al., 2007; Meyers and Prekeris, 2002). In addition to the RBD, each FIP has an α-helical coiled-coil structure mediating FIP homodimerization necessary to execute FIP cellular functions (Wei et al., 2006; 2009; Horgan et al., 2007). FIPs form heterotetrameric complexes with Rab11 composed of two Rab11 and two FIP molecules. Based on their primary structure, FIPs are subcategorized into class I, with FIP1C (alias Rab-coupling protein), FIP2 and FIP5 (alias Rip11, Gaf-1/Gaf-1b, pp75 or gamma-SNAP Associated Factor), and class II, with FIP3 (alias Arfophilin or Eferin) and FIP4 (alias Arfophilin-2). Class I FIPs have a phospholipid-binding C2-domain and are involved in many endosomal recycling and trafficking processes (Lindsy et al., 2002; Lindsay and McCaffrey 2004b; Fan et al., 2004; Prekeris et al., 2000). Class II FIPs contain a calcium binding EF-Hand domain, function in targeting new membranes to the cleavage furrow for the completion of cytokinesis and are necessary for the structural integrity of the pericentrosomal endocytic recycling compartment (ERC) (Horgan et al., 2007). The ERC is constituted of a large complex of heterogeneous subsets of RE, tubular RE and small transport intermediates (Maxfield and McGraw, 2004). FIP3 and FIP4 also bind members of the Arf GTPase family, most notably Arf6, on an Arf-binding domain (ABD) in the C-terminal region that is distinct from the RBD (Shin et al., 1999; Fielding et al., 2005; Shiba et al., 2006). Arf6 forms ternary complexes with Rab11 and FIP3 or FIP4, and mediates the recruitment of Rab11-FIP3 and Rab11-FIP4 to the cleavage furrow (Horgan et al., 2004; Wallace et al., 2002; Wilson et al., 2004; Fielding et al., 2005). Deletion of the ABD from FIP3 in neurons leads to defects in Arf6-regulated delivery of endosome recycling pathways, such as aberrant cytoplasmic retention of N- cadherin and syntaxin 12. The resulting phenotype isa imp irment in neuron migration, which reveals the importance of Arf6-FIP endosomal trafficking pathways (Hara et al., 2016). In this study, we sought to address the specificity of host Rab vesicle interaction with the PV of Toxoplasma by focusing on mammalian Rab11 vesicles. In particular, we sought to examine whether Toxoplasma hijacks indiscriminately any host Rab11A or Rab11B vesicle regardless of composition or bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

localization in infected cells, or if the parasite selectively recognizes subpopulations of Rab11A or Rab11B vesicles associated with specific effectors. Using quantitative microscopy, we assessed the distribution of ectopically expressed, fluorescently tagged mammalian FIP (FIP1C, FIP2, FIP3, FIP4 and FIP5) and Arf6 in Toxoplasma-infected cells. We further determined the contribution of Rab11 or Arf6 binding to the internalization of FIP3 into the PV through mutations introduced in the RBD and/or ABD of FIP3. Our in vitro assays reveal differential interception and PV internalization of host RE based on effector and modulator protein composition. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Results

Mammalian Rab11 vesicle subpopulations with either Class I or Class II FIPs are associated with the PV of Toxoplasma In mammalian cells, the intravacuolar parasite Toxoplasma intercepts the trafficking of host recycling vesicles (Romano et al., 2017). The recruitment of host Rab11 vesicles at the PV was monitored by immunofluorescence assays (IFA) in infected VERO cells using an antibody recognizing Rab11 proteins (Figure 1A). Compared to uninfected cells showing Rab11 puncta near the nucleus and dispersed throughout the cytoplasm, the Rab11 signal in infected cells was observed surrounding the PV (delineated by TgGRA7 staining). The interior of the PV is characterized by the presence of an entangled network of membranous tubules (~30 nm diameter), named the IntraVacuolar Network (IVN) (Sibley et al., 1995). Previously, we illustrated by electron microscopy (EM) the attachment of IVN tubules to the PV membrane providing ‘open gates’ for the entry of host material into the PV, and we detected host organelles, including Rab vesicles within the tubules forming the IVN (Romano et al., 2017). Our IFA on infected cells confirmed the intra-PV distribution of Rab11 vesicles at the IVN stained with TgGRA7 (Figure 1A). In VERO cells expressing GFP-Rab11A or GFP-Rab11B, the fluorescent signal was perinuclear in uninfected cells then redistributed at the PV upon infection, with GFP foci also observed in the PV lumen (Figure 1B and 1C). Confirmation of the GFP signal corresponding to Rab11A in transfected cells was further confirmed by immunostaining with anti-Rab11A antibody, showing significant colocalization of the two fluorescent signals in uninfected VERO cells (Figure 1D, panel a). In transfected and infected VERO cells, the GFP and antibody signals were observed around the PV (Figure 1D, panel b) and within the vacuole (Figure 1D, panel c). Next, we wanted to examine whether Toxoplasma selectively recruits subpopulations of mammalian Rab11 vesicles associated with specific effectors that govern their localization and recycling function. We transiently transfected HeLa cells with a plasmid containing a FIP from Class I (FIP1C, FIP2, FIP5) or Class II (FIP3, FIP4) fused N-terminally with GFP. Post-transfection, cells were infected with RFP-expressing Toxoplasma for 24h. In uninfected HeLa cells, GFP-FIP1C exhibited a wide distribution in the cytoplasm demarcating tubular structures preferentially distributed at the perinuclear region (Figure 2A, panel a). In infected cells, GFP-FIP1C structures were observed concentrated around the PV. In uninfected cells expressing GFP-FIP2, the fluorescent pattern was similar to that of GFP-FIP1C, and upon infection, it also delocalized to the PV (Figure 2A, panel b). Compared to the GFP-FIP1C and GFP-FIP2, the fluorescent signal of GFP-FIP5 was predominantly centralized in the juxtanuclear region of uninfected cells (Figure 2A, panel c). Following infection, GFP-FIP5 localization was more prominent at the PV than at the host nucleus. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Next, we compared the localization of Class II FIPs (FIP3, FIP4) in uninfected and infected cells. It has been reported that exogenous expression of FIP3 or FIP4, which localize to the endocytic recycling compartment (ERC), induces alteration in the morphology of the ERC as this organelle condenses around the microtubule-organizing center (MTOC) at the pericentrosomal region of cells during interphase (Horgan et al., 2004; Wallace et al., 2002). During cytokinesis, FIP3 and FIP4 vesicles move to the cleavage furrow, a process also amplified upon FIP3 and FIP4 overexpression (Horgan et al., 2004; Wallace et al., 2002; Wilson et al., 2005; Fielding et al., 2005). In HeLa cells expressing GFP-FIP3 or GFP-FIP4, we confirmed an intense fluorescent staining at the ERC and the cleavage furrow in non-dividing and mitotic cells, respectively (Figure 2B, panels a and b). Upon Toxoplasma infection, the GFP-FIP3 or GFP-FIP4- positive ERC was delocalized from its pericentrosomal location and associated with the PV.

Toxoplasma internalizes mammalian Rab11 vesicle subpopulations with either Class I or Class II FIPs into the PV with some selectivity Aggregation at the Toxoplasma PV of host vesicles marked with FIP1C, FIP2, FIP5, FIP3 or FIP4 may precede the internalization of these vesicles into the PV. In infected HeLa cells expressing each GFP-FIP protein, the intra-PV localization of GFP-FIP foci was monitored by fluorescence microscopy by collecting a series of optical z-sections throughout the PV defined by TgGRA7 immunostaining. GFP-FIP foci were observed within the PV for all the FIP constructs from Class I (Figure 3A) and Class II (Figure 3B). However, the percentage of PV positive for intra-PV GFP-FIP foci was different among the FIPs; the lowest PV percentage was measured for FIP1C (12%) and the highest for FIP3 (88%) (Figure 3C). Approximately 50% of PV contained FIP2, FIP5 or FIP4 vesicles. A Toxoplasma mutant lacking two-IVN-localized proteins involved in IVN biogenesis (TgGRA2 and TgGRA6) shows complete disruption of this network, which is reduced to loose membrane whorls (Mercier et al., 2002) and is impaired in host organelle sequestration into the PV (Figure S1; Romano et al., 2017). This mutant was used as a negative control in our assays, and as expected, no GFP foci were detected in the vast majority of the Δgra2Δgra6 PV in HeLa cells expressing GFP-FIPs from Class I and Class II (Figure S2).

PV sequestration of vesicles marked with Class I FIP but not Class II FIP requires the formation of Rab11-FIP complexes FIP proteins play a role in targeting Rab11 to different endocytic compartments by competing with each other for Rab11 binding (Meyers and Prekeris, 2002). We next wanted to examine whether the recruitment of FIP-associated vesicles at the PV and their internalization into the vacuole require the presence of Rab11 bound to FIP. Substitution of the hydrophobic isoleucine residue to a negatively charged glutamic acid within the Rab11-binding domain (RBD) of FIP1C (I621E mutation), FIP2 (I481E mutation), FIP5(I630E bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

mutation), and FIP3 (I738E mutation) or replacement of the tripeptidic sequence tyrosine-methionine- aspartate by three alanine residues in the RBD of FIP4 (YMD617-619AAA) totally abrogates FIP binding to Rab11 and the distribution of FIP-RBD mutant vesicles is grossly altered in mammalian cells (Lindsay and McCaffrey 2004a; Junutula et al., 2004; Jagoe et al., 2006; Wilson et al., 2005; Fielding et al., 2005; Horgan et al., 2007; Meyers and Prekeris, 2002). We transiently transfected HeLa cells with plasmids containing each FIP-RBD mutant fused to GFP, and the GFP signal was predominantly dispersed throughout the cell, with a noticeable loss of pericentrosomal ERC staining for FIP3 and FIP4 (Figure 4A and 4B). Following a 24h infection of transfected cells, fluorescent perivacuolar labeling was observed, suggesting the ability of the parasite to recruit FIP-RDB mutant vesicles, even those scattered in the host cytoplasm. Interestingly, many PV in HeLa cells expressing the mutants FIP3-RBD and FIP4-RBD contained numerous GFP foci (Figure 4B). In contrast, no intra-PV GFP signal was detected for the Class I FIP-RBD mutants (Figure 5A). For the Class II mutants, all PV were positive for FIP4-RBD mutant vesicles and three-fourths of PV for FIP3-RBD mutant vesicles (Figure 5B). No GFP foci of FIP3-RBD and FIP4-RBD mutants were spotted in the PV of Δgra2Δgra6 parasites, as expected (Figure S2). The number of intra-PV GFP-FIP foci was counted for all PV in infected HeLa cells expressing Class I and II FIP-WT and Class II FIP-RBD mutants (Figure 6A). Examining the statistical significance for all PV, including ones without intra-PV foci better represents the accuracy of the percent of positive PV counts, thus revealing how often internalization events occur. Focusing on the number of foci only present in positive PV highlights any differences in intra-PV foci abundance; our data in Figure 6B show that the greatest number of intra-PV vesicles was scored for the Class II FIP mutants, with an average number of 7 foci (7.4 FIP3-RBD foci/positive PV) and (6.3 FIP4-RBD foci/positive PV). We conducted immunoEM studies on HeLa cells expressing GFP-FIP3 or GFP-FIP3-RBD mutant using anti-GFP antibody to scrutinize the GFP-positive vesicles around and inside the PV. In uninfected GFP-FIP3-expressing cells, gold particles were detected on the membranes of several vesicles and tubules in the cytoplasm (Figure 7A, panel a). In infected transfected cells, a noticeable gathering of labeled structures was observed around the PV (Figure 7A, panel b). Gold particles were also present on intra-PV vesicles (Figure 7A, panel c), and high magnification observations revealed the gold staining on vesicular membranes (Figure 7A, panel d). In HeLa cells expressing GFP-FIP3-RBD, the gold particle staining was more diffuse throughout the cell and partly associated with vesicles (Figure 7B, panel a). Similar to the GFP-FIP3 WT signal in infected cells, gold particles were observed in close proximity to the PV (Figure 7B, panel b); numerous labeled vesicles were observed in the PV lumen (Figure 7B, panel c), and membrane-associated on vesicles inside IVN tubules (Figure 7B, panel d). Jointly, these observations illustrate a striking difference between the two FIP classes regarding FIP vesicle internalization into the PV as the formation of Rab11-FIP complexes is required for Class I but bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

is dispensable for Class II and even unfavorable based on the greater number of FIP3- and FIP4-RBD mutant vesicles detected in the PV.

The PV contains mammalian vesicles marked with Rab11A, FIP3 or Rab11A-FIP3 complexes The internalization of FIP3-positive vesicles into the PV independently of Rab11 led us to investigate the percentage of intra-PV Rab11A vesicles that are associated with FIP3. Transfected HeLa cells expressing GFP-FIP3 were immunolabeled with anti-Rab11A antibody to assess the colocalization of FIP3 and endogenous Rab11A proteins on vesicles. Expression of FIP3 in HeLa cells resulted in the congregation of Rab11A vesicles on pericentrosomal ERC with FIP3 (Figure 8A, panel a) as compared to non-transfected cells where Rab11A vesicles (detected by immunostaining) distributed throughout the cell with some concentration at the ERC (Figure 1A). The GFP-FIP3 signal was also distributed on ERC tubular extensions that contact Rab11A vesicles. In dividing cells, co-distributed GFP-FIP3 and Rab11A on vesicles were observed close to the intercellular cytoplasmic bridge formed between daughter cells during cytokinesis (Figure 8A, panel b), supporting targeted delivery of recycling endosomal Rab11 vesicles mediated by FIP3 to the midbody (Horgan et al., 2004; Wallace et al., 2002; Wilson et al., 2005; Fielding et al., 2005). Upon infection, the ERC, co-stained for GFP-FIP3 and Rab11A, was delocalized to the PV (Figure 8B, panel a). As illustrated in orthogonal views of z-slices, intra-PV vesicles marked for both FIP3 and Rab11A (Figure 8B, panel b), or only FIP3 or Rab11A were detected in close proximity to each other (Figure 8B, panel c) or distant (Figure 8B, panel d). Of 60 fluorescent foci analyzed from 14 PV, 27% of the foci were yellow (positive for both FIP3 and Rab11A), 50% red (Rab11A only) and 23% were GFP-labeled (FIP3 only). Although our assays could not take into account the presence of endogenous FIP3 associated with intra-PV vesicles, the detection of vesicles with GFP-FIP3 (excluding endogenous Rab11A) suggests the heterogenous composition of host recycling vesicles sequestered inyo the PV.

Mammalian FIP3 vesicles are internalized in the PV in association with Arf6 The Rab11A-independent sequestration of FIP3 and FIP4 vesicles into the PV prompted us to investigate whether other FIP interactors were associated with Class II FIP vesicles in the PV lumen. Both FIP3 and FIP4 are Arf6 effectors (Shin et al., 2001). In mammalian cells, Arf6 localizes to the plasma membrane and endosomal compartments, and it is involved in the targeted delivery of recycling endosomal vesicles to the plasma membrane (D’Souza-Schorey and Chavrier, 2006). In preparation for cell division, Arf6 plays a vital role in targeting RE, e.g., Rab11 to the cleavage furrow and midbody (Schweitzer and D’Souza- Schorey, 2002). To compare the distribution of Arf6 in uninfected and Toxoplasma-infected cells, HeLa cells were transiently transfected with a plasmid containing Arf6 in fusion with C-terminal mCherry. In uninfected cells, the Arf6-mCherry signal was distributed in cytoplasmic structures with a higher bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

concentration at the perinuclear region (Figure 9A). When transfected cells were infected for 24h, a strong perivacuolar staining was observed for Arf6-mCherry, suggesting interaction of the Toxoplasma PV with Arf6-positive vesicles (Figure 9B, panels a and b). Several Arf6-mCherry foci were observed within the PV, often concentrated at the IVN (Figure 9B, panel b). To examine whether the parasite recognizes vesicles with both Arf6 and FIP3, we transiently transfected HeLa cells with two plasmids, one encoding GFP-FIP3 and the other Arf6-mCherry. Previous studies reported that exogenously co-expressing FIP3 and Arf6 results in increased colocalization of the two proteins (Horgan et al., 2004; Fielding et al., 2005). In dually transfected cells, we confirmed an intense co-labeling of pericentrosomal ERC with GFP-FIP3 and Arf6-mCherry (Figure 9C, panel a). In dividing cells, the co-migration of vesicles positive for FIP3 and Arf6 was observed at the intersection between the two daughter cells, as illustrated in panel b in Figure 9C showing midbody remnants at the cell surface after abscission. In infected co-transfected cells, the Arf6-mCherry and GFP-FIP3 signals largely overlapped at the ERC close to the PV membrane and vesicles with Arf6 and FIP3 were observed within the PV (Figure 9D). In two independent assays, 86% and 61% of PV contained Arf6-mCherry vesicles upon mono- transfection or double transfection, respectively (Figure 10A). Approximately 70% of the Arf6-mCherry foci colocalized with the GFP-FIP3 foci were positive for Arf6-mCherry in cells expressing both Arf6 and FIP3. To determine whether Arf6 binding to FIP3 facilitates the Rab11-independent entry of class II FIP vesicles into the PV, we co-transfected HeLa cells with plasmids encoding Arf6-mCherry and GFP-FIP3- RBD mutant. The Arf6-mCherry and GFP-FIP3-RBD signals were dispersed in uninfected and infected cell, with partial colocalization (Figure 10B). The perivacuolar staining for Arf6-mCherry and GFP-FIP3- RBD was weaker in dually transfected and infected cells (Figure 10B) than for infected HeLa cells transfected with Arf6-mCherry alone (Figure 9B), GFP-FIP3 alone (Figure 2B, panel a), or Arf6-mCherry and GFP-FIP3 (Figure 9D). In these two independent assays, only ~23% of PV contained Arf6-mCherry foci, and less than 30% of the Arf6-mCherry foci colocalized with the GFP-FIP3-RBD foci. There were also significantly less Arf6-mCherry foci per PV when co-transfected with GFP-FIP3-RBD as compared to co-transfection with GFP-FIP3 (Figure 11A). In comparison to the number of Arf6-mCherry foci per PV, the number of GFP-FIP3 or GFP-FIP3-RBD number of foci per PV remained constant upon double transfection. In fact, the number of GFP-FIP3 foci per PV increased following co-transfection with Arf6- mCherry while the number of Arf6-mCherry foci per PV dropped (Figure 11B). These data suggest that the presence of Rab11 complexed to FIP3 leads to more efficient internalization of Arf6 vesicles into the PV and that Arf6-FIP3 interactions may not be the driving force for the delivery of vesicles of class II FIP-RBD mutants to the PV. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Binding of FIP3 to either Rab11 or Arf6 increases the delivery of mammalian vesicles displaying FIP3 into the PV FIP3 binds to the C-terminal α-helix of Arf6 (Schonteich et al., 2007). We generated a GFP-FIP3 construct with an altered ABD through the truncation of residues 687 to 712 to investigate further the relevance of FIP3 complexed to Arf6 for recognition and internalization of vesicles into the PV. HeLa cells were transfected with a plasmid encoding GFP-FIP3-ABD (FIP3Δ687-712) or a plasmid encoding the dual mutant GFP-FIP3-RBD-ABD (FIP3I738EΔ687-712), infected and analyzed by fluorescence microscopy. Numerous foci for GFP-FIP3-ABD vesicles were observed in the PV interior (Figure 12A) while no GFP-FIP3-RBD-ABD vesicles were detected in the vast majority of PV (Figure 12B, panel a). For PV in which GFP-FIP3-RBD- ABD foci were observable, the GFP signal was very weak and the foci were tiny (Figure 12B, panel b) compared to foci for GFP-FIP3 WT or GFP-FIP3-RBD. The percentage of PV containing vesicles with GFP-FIP3-ABD and the number of those vesicles as quantified in Figure 12C were comparable to the percentage of PV and number of vesicles for GFP-FIP3 WT or GFP-FIP3-RBD vesicles (Figures 3C, 5C and 6). However, only 37% of PV were positive for the dual mutant GFP-FIP3-RBD-ABD, and the intra- PV foci for GFP-FIP3-RBD-ABD were very tiny. These data suggest that Rab11-FIP3 or Arf6-FIP3 interactions may not be individually require to drive FIP3 vesicle sequestration into the PV but abrogating the binding of both Rab11 and Arf6 to FIP3 is detrimental for this process. However, since abrogation of both binding processes does not completely impede vesicle delivery to the PV, other FIP3 interactors may drive the internalization. A graphical summary of our data presented in Figure 13 illustrate the Class I FIP dependence on Rab11 binding for internalization and the PV’s ability to internalize FIP3 vesicles depending on whether their interaction with Rab11, Arf6, or neither.

Toxoplasma infection results in the altered expression of several genes involved in the host mammalian recycling system The intersection of the Toxoplasma PV with mammalian Rab11, FIPs and Arf6 proteins that regulate recycling endocytosis or cytokinesis led us to examine more broadly the impact of infection on the host recycling endosomal network. We performed RNA sequencing analysis on Toxoplasma-infected fibroblasts 24h post-infection to survey alterations in the expression of genes encoding Rabs, Rab GTPase activating proteins (RabGAP) and Rab guanine nucleotide exchange factors (RabGEF), which control the recycling system (Figure 14; see details of all genes in Table SI and description some effectors in Figure S3). Among Rab proteins, Rab15, which co-localizes with Rab4, Rab5 and the TfR on sorting endosomes (SE) as well as with Rab11 on the ERC (Zuk and Elferink, 1999; 2000), is up-regulated upon infection. Rab15 regulates the TfR trafficking through SE and the ERC, and influences iron levels in mammalian cells. Concerning bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Rab5 proteins, the Rab5b isoform, which localizes to a TfR-positive endosomal compartment (Wilson and Wilson, 1992; Bucci et al., 1995), is down-regulated in infected cells. Upon Toxoplasma infection, no changes in expression levels were detected for other Rabs involved in recycling such as Rab11 and Rab35; Rab35 is a major player in the fast recycling pathway and cytokinesis and localizes at the plasma membrane and endosomes (Klinkert and Echard, 2016). TBC domain-containing proteins (TBC proteins) function as RabGAP, promoting the hydrolysis of Rab-GTP to Rab-GDP for the regulation of specific intracellular trafficking pathways. TBC1D4 enhances glucose uptake in cells through glucose transporter 4 (GLUT4) (Kane et al., 2020; Miinea et al., 2005; Sano et al., 2003; Treeback et al., 2010) and is up-regulated in infected cells. TBC1D11, which is down-regulated in infected cells, is involved in cytokinesis through Arf6-positive RE recruitment (Kanno et al., 2010; Kobayashi et al., 2014). The Rab11 GAP EVI5 is also down-regulated in infected cells, is a modulator of cell cycle progression (Lim and Tang, 2013), and co-localizes with TBC1D11 in RE containing TfR (Kanno et al., 2010). After prenylation, Rab proteins are delivered to a target membrane where they are activated by a GEF to replace bound GDP by GTP. The Rab8 GEF Rabin-8 is up-regulated in infected cells and it controls the exocytic trafficking of proteins from the TGN and RE vesicles containing TfR to the plasma membrane (Feng et al., 2015) and co-distributes with Arf6 for vesicle delivery to the plasma membrane (Hattula et al., 2006; Peranen, 2011; Feng et al., 2015). GRAB (RAB31L1) is down-regulated in infected cells and is implicated in the exo-endocytic cycling of vesicles (Pavlos et al., 2010). Mss4 (RabIF) is up-regulated upon infection, and this holdase chaperone promotes the stability of Rab10, a key Rab in GLUT4 exocytosis (Gulbranson et al., 2017). Factors for Endosome Recycling And Rab Interactions (FERARI) is a tethering platform for multiple effectors acting in Rab11 recycling pathways at SE (Solinger et al., 2020). The Rab-binding module of FERARI consists of FIP5 (binds to Rab11) and rabenosyn-5 (binds to Rab4 and Rab5), while the SNARE-interacting module comprises VPS45 and VIPAS39 (de Renzis et al., 2002; Solinger et al., 2020; van der Kant et al., 2015; Mohrmann et al., 2002). All four of these components from FERARI are down-regulated in infected cells. Endocytosed transferrin is sequentially transported through Rab5, Rab4 and Rab11 vesicles before recycling back to the plasma membrane (Sonnichsen et al., 2000; Ullrich et al., 1996; Daro et al., 1996). Rabenosyn-5 stimulates TfR recycling by activating the fast pathway and switching off the slow recycling route through Rab11-positive membranes (Mohrmann et al., 2002). FERARI, which also promotes membrane fission and fusion of SE at the ERC, includes the dynamin-like protein EHD1 (Daumke et al., 2007) that is up-regulated upon Toxoplasma infection. EHD1 exerts its pinching activity on tubular endosomal membranes, playing a role in ERC maintenance (Solinger et al., 2020). bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The TRAnsport Protein Particle (TRAPP) is a modular multiunit complex that acts as a GEF for Rab proteins and thus is an important regulator of membrane traffic (Lipatova et al., 2019; Sacher et al., 2019). Upon infection, four TRAPP components have dysregulated expression, including TRAPPC1, TRAPPC6A, and TRAPPC3L that are part of the TRAPP core (Kim et al., 2016). Kalirin, which is one of the four subunits forming a core that confers the GEF activity onto TRAPP, forms a complex with several TRAPP, including TRAPPC9 to facilitate the recycling activities of Rab11 such as for TfR (Kim et al., 2006; Wang et al., 2020). Of the 5 TRAPP components with dysregulated expression, 4 out 5 are down- regulated, which suggests decreased preference for the slow Rab11 recycling of TfR. Overall, several genes encoding effectors involved in recycling pathways (e.g., for GLUT4 and TfR) have differentiated expression in Toxoplasma-infected cells compared to uninfected cells, suggesting the manipulation of many host recycling compartments and effectors by the parasite, perhaps to increase nutrient (e.g., glucose, iron) pools in the host cell. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion

In this study, we reported that the intravacuolar parasite Toxoplasma manipulates the trafficking pathways of mammalian recycling endosomes (RE) that contain lipids as cargoes destined for the plasma membrane for re-use by the cell or for membrane donation to form the cleavage furrow during cytokinesis. Mammalian Rab11 proteins regulate the traffic of the slow recycling pathway for many cell surface proteins, including TfR, ion channels, junctional proteins, integrins, and immune receptors (Campa & Hirsch, 2017). We have shown that Rab11A and Rab11B vesicles distribute around the PV and are internalized into the vacuole (Romano et al., 2017), and that subsets of Rab11 vesicles with Class I and class II FIP are differentially scavenged by Toxoplasma (this study). A previous study showed that ectopically expressed Arf6 surrounds the PV in infected VERO cells during invasion, which potentially facilitates PV membrane biogenesis (da Silva et al., 2009). Here, we demonstrated that the perivacuolar localization of mammalian Arf6 persists throughout infection and that Arf6 vesicles penetrate the PV. Finally, we observed that the ERC is dramatically delocalized from its pericentrosomal region of the host cell to be associated with the PV, underlying important alterations in the recycling system in the host cell.

Why does Toxoplasma divert host Class I FIP vesicles? Class I FIPs function in endosomal recycling processes (reviewed in Jing and Prekeris, 2009) and bind to Rab11 to govern the trafficking of Rab11 vesicles to the cell surface. The C2-domain of class I FIPs targets the Rab11-FIP complexes to docking sites on the plasma membrane selectively enriched in phosphatidylinositol-3,4,5-trisphosphate and phosphatidic acid, two lipids required for fusion (Lindsay and McCaffrey, 2004b). Toxoplasma may advantageously intercept Class I FIP vesicles to scavenge their cargo for nutritional benefit and/or to neutralize toxic host factors through their sequestration into the PV. Among specific cargo trafficked to the cell surface by Class I FIP, the glucose transporter GLUT4 is trafficked to the cell surface via FIP5, which partially localizes to intracellular GLUT4 storage vesicles (GSV) (Welsh et al., 2007). FIP1C mediates the recycling of several plasma membrane receptors, including TfR, and FIP2 is implicated in the recycling of many molecules, e.g., GLUT4, TfR, the water channel protein aquaporin-2 and the chemokine receptor CXCR2 (Horgan and McCaffrey, 2009). The cargo of Class I FIP vesicles may supply nutrients for replicating Toxoplasma. For example, the source of iron for Toxoplasma is still unknown, and FIP1 and FIP2 vesicles associated with TfR-Tf may provide iron to the parasite. Of interest, our RNA seq data reveal that genes coding for regulators of TfR recycling, such as TBC1D11, EVI5, Rabenosyn-5 and Kalirin, have altered expression upon infection and these changes may affect the TfR cycle leading to the retention of iron-loaded transferrin in infected cells to the parasite’s profit. GLUT4 is the main glucose transporter in muscle and fat cells, and it localizes at bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

the plasma membrane and many organelles such as SE, RE and the TGN as well as GSV that mediate the transport of GLUT4 between these compartments (Bryant et al., 2002). In the absence of insulin, about ~35% of GLUT4 remain trapped in an intracellular circuit between endosomes and the TGN (Martin at al., 1996). In fibroblasts (used in our RNAseq screen), most GLUT4 localizes with TfR in endosomes (Lampson et al., 2001). TBC1D4 that shuttles GLUT4 between GSV and the plasma membrane, is up- regulated in Toxoplasma-infected cells, potentially leading to increased glucose intake, which may be beneficial for the parasite. Moreover, the RabGEF Mss4 that stabilizes Rab10, a GTPase involved in GLUT4 exocytosis and, thus mediating GLUT4 surface-exposure, is up-regulated upon infection. In skeletal muscle cells, GLUT4 is highly expressed to promote glucose intake in response to insulin for energy demands. The tropism and attraction of Toxoplasma for skeletal muscle cells for encystation (Swierzy and Lüder, 2015) may be linked to the metabolic properties of skeletal muscle cells, such as a high glucose supply, which represent a propitious environment for the long-term survival of the parasitic chronic stage. Toxoplasma may also target Class I FIP vesicles to modulate the immune response of its host. For example, the chemokine receptor CXCR2 is present into Rab11A-positive RE (Fan et al., 2003), and its recycling and thus receptor-mediated chemotaxis is regulated by FIP2 (Fan et al., 2004). CXCR2 stimulates cell migration in response to a concentration gradient of the CXC chemokine IL-8 ligand (Wolf et al., 1998), and is essential for the recruitment of leukocytes to inflammatory sites (Garcia-Ramallo et al., 2002). Neutrophils employing a battery of microbicidal activities, e.g., phagocytosis, neutrophil extracellular trap (NET) formation and release of peroxide species, are required for early resistance against Toxoplasma in the gut (Sayles and Johnson, 1997; Bliss et al., 2001). In particular, CXCR2 is involved in early neutrophil recruitment and plays an important protective role in resistance to Toxoplasma (Del Rio et al., 2001). In fact, Toxoplasma infection of CXCR2−/− mice results in defects in neutrophil migration to the site of infection, lower production of proinflammatory cytokines (e.g., TNF-α, IFN-γ), and higher brain cyst numbers during chronic infection, compared to WT mice. To this point, the sequestration of vesicles containing CXCR2-FIP2 complexes into the PV may be advantageous for the parasite to avoid neutrophil attraction to infected tissues, thus ensuring parasite dissemination in the host. In addition to CXCR2, the cargoes of Rab11 vesicles include immune mediators (Kelly et al., 2012): TNF-α, which triggers IFN-γ-primed macrophages for Toxoplasma killing (Sibley et al., 1991); IL- 6, which protects during Toxoplasma early infection by promoting IL-17 production from NK cells for T- cell activation (Jebbari et al., 1998; Passos et al., 2010) and TLR4, which is a critical innate immune cell receptor involved in Toxoplasma detection and activation of immune responses (Zare-Bidaki et al., 2014). Diversion of these anti-Toxoplasma effectors to the PV could be part of a strategy crafted by the parasite to thwart host cell-intrinsic innate immunity pathways. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The internalization of Class I FIP vesicles is Rab11-dependent, suggesting the recognition of complexes formed between FIP1, FIP2 or FIP5 and Rab11. This would ensure an elective and efficient targeting of Class I FIP-Rab11 RE by the parasite to benefit from their cargoes.

Why does Toxoplasma divert host Class II FIP vesicles? While class I FIP are specifically implicated in the regulation of endocytic protein sorting and recycling, class II FIP have different roles and more specialized functions depending on the cell cycle (reviewed in Jing and Prekeris, 2009). FIP3 and FIP4 localize to the ERC and act as scaffold proteins for this organelle, tethering the ERC to the pericentrosomal region (in many cell types) through binding to microtubule motor proteins. Knock-down of FIP3, overexpression of FIP4 or alteration of the microtubule cytoskeleton (e.g., with nocodazole) causes abnormal distribution of the ERC to the cell periphery (Horgan et al., 2007; Wallace et al., 2002; Lin et al., 2002). In Toxoplasma-infected cells, the host ERC is dragged toward the PV. This process may be consecutive to the hijacking of the host MTOC by the parasite, resulting in the rearrangement of the host microtubular network all around the vacuole (Coppens et al., 2006; Walker et al., 2008). Manipulating the host microtubular network allows for the parasite to govern the host cellular membrane transport and attract organelles to the PV. Of importance, the recruitment of the host MTOC and microtubules is actively mediated by the parasite as these events also occur in mammalian cytoplasts that lack genetic materials and are, in other words, dying fragments of cytoplasm (Romano et al,. 2008). The co-option of host microtubules is also associated with supranumerous foci of γ-tubulin and aberrant multi-nucleated structures in infected cells, leading to cell cycle dysregulation and defects in cytokinetic events. By stalling host cellular division and blocking cytokinesis, the parasite may insure a vast and favorable environment for replication by increasing resource availability, such as lipid membranes that would normally be targeted to the cleavage furrow to complete cytokinesis. During cytokinesis, FIP3 or FIP4 associated with Rab11 moves Rab11 RE to the cleavage furrow via centrosome-anchored microtubules (Fielding et al., 2005). At the furrow, the Rab11-FIP3 or Rab11-FIP4 vesicles encounter and interact with Arf6 on the plasma membrane, resulting in tethering of these vesicles to the plasma membrane via interaction with the Exocyst complex. In this case, the attraction and retention of FIP3, FIP4 and Arf6 vesicles in the PV may represent an additional stratagem developed by Toxoplasma to cause cytokinesis failure by blocking the abscission process. Alternatively, the primary target of Toxoplasma may be the host ERC, which is pulled toward the PV subsequently to MTOC capture. The ERC is a long-lived compartment, with multiple sorting functions toward the recycling, retrograde and exocytic routes (Maxfield and McGraw, 2004). Most proteins that cycle between the cell interior and the cell surface accumulate in the ERC because this is the slow step in their return to the plasma membrane. For example, all of the LDL receptors and TfR recycle from the ERC bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

to the cell surface. Rab15 regulates iron levels in the ERC (Zuk and Elferink, 1999; 2000); Rab15 up- regulation in infected cells may result in an increase in iron content in the ERC. The ERC is also a major repository of cholesterol that enters the organelle by endocytosis and non-vesicular mechanisms (Hao et al, 2002). A prime example of essential metabolites for the parasite is cholesterol, which is derived from LDL that are internalized into the host cell (Coppens et al., 2000). Toxoplasma also relies on host sphingolipids for optimal replication (Romano et al., 2013), and sphingomyelin transits through the ERC prior to delivery to various intracellular destinations (Koval and Pagano, 1989). The ERC may then represent a source of endogenous and exogenous metabolites for Toxoplasma, and targeting the ERC for its massive pool of cholesterol and sphingolipids would be advantageous for the parasite. The proximity of the ERC to the PV would also facilitate the scavenging of FIP3 and FIP4 vesicles by the parasite. The ERC is composed of both Rab11- and Arf6-positive membrane subsets. The trapping of FIP3 and FIP4 vesicles inside the PV is Rab11-independent, suggesting a mechanism of internalization for class II FIP vesicles different from Class I FIP vesicles, such as the mediation of Arf6 for recognition and scavenging of class II FIP vesicles. Overall, our data highlight a selective process of interaction with host vesicular components at the PV membrane, likely mediated by specific parasite proteins. Arf6 mediates a clathrin-independent endocytosis (CIE) pathway, in which cargo is internalized and transported in Arf6-enriched vesicles to be subsequently recycled to the plasma membrane (Van Acker et al., 2019). Thus, like Rab11-associated vesicles, Arf6-associated endosomes may play roles in nutrient acquisition or immune response control. Arf6-associated endosomes carry cholesterol and Arf6 is a central regulator of cholesterol homeostasis, controlling both the uptake and efflux of this lipid from cells (Naslavsky et al., 2004; Schweitzer et al., 2009). Therefore, the intra-PV Arf6 vesicles may constitute a providential source of cholesterol for Toxoplasma. Additional potential nutrient modulating cargo for Arf6- associated endosomes include the glucose transporter GLUT1 and some amino acid transporters. MHC class I molecules are also present on Arf6-positive tubules emanating from the ERC. It has been reported that cross‐presentation by MHC class I molecules, which allow the detection of exogenous antigens by CD8+ T lymphocytes, is crucial to initiate cytotoxic immune responses against Toxoplasma (Blanchard et al., 2008). Interestingly, the Δgra2Δgra6 Toxoplasma mutant that lacks the IVN and is impaired in intra- PV vesicle internalization, is more susceptible to MHC I presentation than WT parasites (Lopez et al., 2015). This suggests that the scavenging of Arf6 vesicles could be an immune regulatory process mediated by the parasite to interfere with antigen cross‐presentation and immune detection.

Like Toxoplasma, several intracellular bacteria target the mammalian host endocytic recycling circuitry (Brumell and Scidmore, 2007; Allgood and Neunuebel, 2018). By usurping host recycling Rab (e.g., Rab4, Rab11, Rab35), bacteria promote their entry and exit from the host cell, control the biogenesis, bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

maturation or rupture of their vacuole, and evade immune detection. From a therapeutic point-of-view, any essential parasite and bacterial effectors located at the vacuolar membrane or secreted into the host cell for interaction with specific Rab proteins could represent potential drug targets to combat microbial infections. Seminal information on the role of mammalian host Rab effectors has come from work on intracellular pathogens that co-opt Rab functions (Stein et al., 2012; Sherwood and Roy, 2013; Damiani et al., 2014). From a cell biology perceptive, studying the unidirectional transit of host Rab vesicles toward the Toxoplasma PV may reveal which Rab effectors bind to parasite proteins at the PV, perhaps expanding our repertoire of Rab-interacting proteins. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Materials and Methods

Reagents and antibodies - All reagents were purchased from Sigma (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA), unless otherwise stated. The primary antibodies used in this study were: rat and rabbit polyclonal anti-GRA7 (Coppens et al., 2006), rabbit anti-Rab11 (Cell Signaling Technologies, Danvers, MA), mouse anti-NTPase (gift from JF Dubremetz, University of Montpellier, France) and rabbit anti-Rab11A (Cell Signaling Technologies, cat #2413S). This anti-Rab11A antibody was selected to specifically recognize human Rab11A (excluding human Rab11B and Toxoplasma Rab11) based on unique sequence at the C-terminus from amino acid 181 to 216 in human Rab11A. Secondary antibodies from Thermo Fisher Scientific included anti-rat conjugated to Alexa Fluor 350, Alexa Fluor 488 or Alexa Fluor 594, and anti-rabbit conjugated to Alexa Fluor 594 or Alexa Fluor 647.

Cell and parasite cultivation - Vero cells, HeLa cells, and human foreskin fibroblasts (HFF) were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in αMEM with 10% (v/v) FBS, 100 U/mL penicillin/streptomycin (Quality Biological Inc, Gaithersburg, MD) and 2 mM L-glutamine o (complete αMEM culture medium) at 37 C and 5% CO2. The Vero cells stably expressing GFP-Rab11A (Romano et al., 23017) was cultivated in complete αMEM culture medium containing 800 µg/mL G418. Toxoplasma tachyzoites (type 1 RH), WT and transgenic strains, were serially passaged in 6-well plates on confluent monolayers of HFF by transferring the supernatant containing egressed tachyzoites from one well to another (Roos et al., 1995). The RFP expressing RH strain was provided by F. Dzierszinski (McGill University, Montreal, Canada; Dzierszinski et al., 2004). The RH strain with gra2 and gra6 knocked out (Δgra2Δgra6) was provided by MF Cesbron-Delauw (Université Grenoble Alpes, Grenoble, France; Mercier et al., 2002).

Toxoplasma infection of mammalian cells - Transfected cells were infected 4 h following transfection and untransfected cells were infected approximately 24-48 hours after plating at 60% confluency. For infection, media was removed from the cells on coverslips in wells from a 24-well plate, and freshly egressed parasites were added for 30 min. The coverslips were then washed with prewarmed PBS to remove extracellular (non-invading) parasites allowing for synchronization of infection. The infections proceeded for 24 h to yield 4 to 8 parasites per PV.

Plasmids - The plasmids GFP-FIP1C, GFP-FIP1CI621E, GFP-FIP2, GFP-FIP2I480E, GFP-FIP3, GFP- FIP3I738E, GFP-FIP4, GFP-FIP4YMD617-619AAA, GFP-FIP5 and GFP-FIP5I630E were generously provided by M. McCaffrey (University College Cork, Cork, Ireland). Their sequences were verified using the standard bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

CMV-For primer and our primer oJR81 5’ GGGAGGTGTGGGAGGTTTT 3’. The Arf6-mCherry plasmid pcDNA3/hArf6(WT)-mCherry was a gift from Kazuhisa Nakayama (Addgene plasmid # 79422; http://n2t.net/addgene:79422; RRID:Addgene_79422; Makyio et al., 2012) and the sequence was confirmed using standard CMV-For and BGH-Rev primers. The plasmids containing the Arf6-binding domain(ABD) deletion mutant (GFP-FIP3-ABD) and the ABD and Rab11-binding domain (RBD) mutant (GFP-FIP3-I738E-ABD) were engineered from the McCaffrey plasmids GFP-FIP3 and GFP-FIP3I738E using the Q5 Mutagenesis Kit (New England Biolabs, Ipswich, MA) following manufacturer instructions. To generate FIP3-ABD mutant, amino acids 687-712 were deleted using primers ABDdlt_F 5' AGCTCCGTCTCCCGAGAT 3' and ABDdlt_R 5' CCCGTTCAGCTCCTCGTT 3'. The same pair of primers was used for both pEGFP-c1-FIP3 and pEGFP-c1-FIP3 2-756 I738E using an annealing temperature of 68oC with 3% DMSO added to the PCR reaction. Transformed bacteria were grown on LB- agar plates containing kanamycin overnight and then cultured in 3ml LB Broth with kanamycin for 22 h at 37oC at 220 rpm. After transformation and overnight culture, the Qiaprep Spin Miniprep Kit (Qiagen, Germantown, MD) was used to isolate the plasmid, and Sanger sequencing using primer oJR81 was used to confirm the sequence of the FIP3-ABD and FIP3-RBD-ABD mutant plasmids.

Mammalian cell transfection - Vero cells were transfected with 2 µg plasmid DNA in the Amaxa Nucleofector solution R (Lonza Bioscience, Rockville, MD), following the manufacturer’s instructions with program V-01. Transfected Vero cells were then plated on coverslips in 24-well plates and allowed to recover for 24 h before infection or fixation. HeLa cells were plated on coverslips in 24-well plates at the confluency of 45-50% 24-48 h before transfection using jetPrime (Polyplus-transfection, New York, NY) for 4 h following manufacturer’s instructions. Recommended DNA and reagents for HeLa cells were adjusted to 0.2 µg plasmid DNA, 0.4 µL jetPrime reagent and 50 µL jetPrime buffer per well to minimize cell toxicity.

Transcriptome sequencing - Toxoplasma-infected HFF for 24 h and uninfected HFF were PBS-washed, detached with trypsin, collected by centrifugation, and resuspended in PBS buffer. Total RNA from infected and uninfected HFF from 3 independent preparations was extracted using a Qiagen RNeasy kit. The total RNA was shipped to Novogene Corporation (Sacramento, CA) to be processed and analyzed. First, total RNA was converted to sequencing read libraries using the NEB Next Ultra kit (Illumina). The libraries were subjected to paired-end sequencing using the Novaseq 6000 with a read length of 150 bp. Each sample was sequenced to a depth of at least 20 million reads. The sequencing reads per sample were trimmed and mapped to the human genome (Homo sapiens GRCh38/hg38). Differential gene expression analysis was bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

performed using DESeq R (1.18.0) and p-values were adjusted using the Benjamini and Hochberg’s approach for controlling false discovery rate (p < 0.05 considered as differentially expressed).

Immunofluorescence Assay (IFA) - Cells were fixed in solution of 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA) and 0.02% glutaraldehyde (EMS) for 15 min at room temperature and permeabilized with 0.3% Triton X-100 for 5 minutes at room temperature. Cells were blocked with 3% Bovine Serum Albumin (BSA) for 1 h at room temperature before addition of the primary antibody diluted in 3% BSA for 1 h at room temperature or overnight at 4oC. Secondary antibodies diluted in 3% FBS were added to the coverslip for 1 h incubation at room temperature. To stain nuclei, coverslips were incubated with DAPI at a 1 µg/ml in water for 5 min at room temperature. Coverslips were then mounted with the Prolong Gold or Diamond antifade mounting solution.

Fixed cell imaging and analysis - Cells were imaged on a Zeiss Axioimager M2 fluorescent microscope with a z motor for acquisition of slices along the z-axis. An oil-immersion Zeiss plan Apo 100x/NA 1.4 objective and Hamamatsu ORCA-R2 camera were used to acquire images with optical z-slices of 0.2 µm. Images were acquired, registry corrected, deconvolved, and brightness/contrast adjusted using the Volocity imaging software version 6.3 or 6.3.1 (Perkin Elmer, Waltham, MA). Fluorescent foci in the PV were identified and counted manually after identifying the boundaries of the PV based on the change in fluorescent patterns of the fluorescently tagged proteins. Statistical differences were determined using unpaired, two-tailed T-tests with the Welch’s correction for unequal standard deviations with Prism (Graphpad, San Diego, CA). PV were ranked as positive based on one or more distinct fluorescent foci detected within the bounds of the PV or negative if no foci could be observed inside the PV.

ImmunoElectron microscopy - For immunogold staining of GFP-FIP3 and GFP-FIP3-RBD, Toxoplasma- infected cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 0.25 M HEPES (ph7.4) for 1 h at room temperature, and then in 8% paraformaldehyde in the same buffer overnight at 4oC. They were infiltrated, frozen and sectioned as described (Romano et al., 2017). The sections were immunolabeled with antibodies against GFP (Clontech Laboratories Inc, Mountain View, CA) at 1:25 diluted in PBS/1% fish skin gelatin, and then with secondary IgG antibodies coupled to 10 nm protein A- gold particles before examination with a Philips CM120 EM (Eindhoven, the Netherlands) under 80 kV. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Acknowledgements

We thank the members of the Coppens’ laboratory for helpful discussion during the course of this work and especially Karen Ehrenman for her help for the cloning of the ABD mutant. We are also grateful to the generous providers of antibodies, plasmids and parasite strains used in this study. We thank the excellent technical staff and Kim Zichichi of the Electron Microscopy Core Facility at Yale School of Medicine. This study was supported by the grant from the NIH AI060767 to I.C. and a pre-doctoral fellowship from the American Heart Association to E.J.H. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Figure legends

Figure 1. Distribution of mammalian Rab11 vesicles in Toxoplasma-infected cells A. Fluorescence microscopy of VERO cells, uninfected or infected with RFP-expressing Toxoplasma (RFP-Tg) for 24h, before immunostaining with anti-Rab11 and anti-TgGRA7 (PVM/IVN) antibodies. For all images, individual z-slices and orthogonal views highlighting the PV, are shown. B-C. Fluorescence microscopy of VERO cells expressing GFP-Rab11A (in B) or GFP-Rab11B- (in C), uninfected or infected with RFP-expressing Toxoplasma for 24h, before immunostaining with anti-TgGRA7 (PVM/IVN) antibody. D. Fluorescence microscopy of VERO cells expressing GFP-Rab11A-, uninfected (panel a) or infected with Toxoplasma for 24h, before immunostaining with anti-Rab11A antibody, showing colocalization of the Rab11A signals surrounding the PV (panel b) and inside the PV (panel c).

Figure 2. Distribution of mammalian vesicles containing FIP from class I and II in Toxoplasma-infected cells A-B. Fluorescence microscopy of HeLa cells transfected with Class I FIPs (GFP-FIP1C, GFP-FIP2 or GFP- FIP5 in A) and Class II FIPs (GFP-FIP3 or GFP-FIP4 in B), uninfected or infected with RFP-expressing Toxoplasma for 24h. Phase contrast overlaid with the GFP-FIP signal is shown as well as the GFP-FIP alone and with the RFP signal.

Figure 3. Distribution of mammalian vesicles containing FIP from class I and II within the Toxoplasma PV A-B. Fluorescence microscopy of HeLa cells transfected with GFP-FIP (Class I in A: GFP-FIP1C, GFP- FIP2 or GFP-FIP5; Class II in B: GFP-FIP3 or GFP-FIP4), uninfected or infected with RFP-expressing Toxoplasma for 24h before immunostaining with anti-TgGRA7 antibody. DAPI in blue. For all images, individual z-slices and orthogonal views are shown. Arrows show intra-PV GFP-FIP foci. C. Quantification of the internalization of Class I and II FIP vesicles within the Toxoplasma PV. Percent of PV with intra-PV foci was assessed based on PV viewed in A and B. Negative controls include PV from Δgra2Δgra6 mutants impaired in host vesicle internalization.

Figure 4. Distribution of mammalian vesicles containing FIP-RBD mutants from class I and II in Toxoplasma-infected cells A-B. Fluorescence microscopy of HeLa cells transfected with FIP-RBD (Class I in A: GFP-FIP1CI621E, GFP-FIP2I481E or GFP-FIP5I630E; Class II in B: GFP-FIP3I738E or GFP-FIP4YMD617-619AAA), uninfected or infected with RFP-expressing Toxoplasma for 24h. Phase contrast overlaid with the GFP-FIP signal is shown as well as the GFP-FIP alone and with the RFP signal.

Figure 5. Distribution of mammalian vesicles containing FIP-RBD mutants from Class I and II in the Toxoplasma PV A-B. Fluorescence microscopy of HeLa cells transfected with FIP-RBD mutants (Class I in A: GFP- FIP1CI621E, GFP-FIP2I481E or GFP-FIP5I630E; Class II in B: GFP-FIP3I738E or GFP-FIP4YMD617-619AAA), uninfected or infected with RFP-expressing Toxoplasma for 24h before immunostaining with anti-TgGRA7 antibody. DAPI in blue. For all images, individual z-slices and orthogonal views are shown. Arrows show intra-PV GFP-FIP foci. C. Quantification of the internalization of Class I and II FIP-RBD mutant vesicles bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

within the Toxoplasma PV. Percent of PV with intra-PV foci was assessed based on PV viewed in A and B.

Figure 6. Enumeration of Rab11-FIP or FIP-RBD mutant vesicles within the Toxoplasma PV Intra-PV vesicles containing FIP or FIP-RBD mutants from class I and II were counted based on GFP foci in all the PV (A) or only in GFP-positive PV (B). Two-tailed p values shown. ns, p >0.05.

Figure 7. Immunolocalization of FIP3 and FIP3-RBD mutant in Toxoplasma-infected cells A-B. ImmunoEM of HeLa cells expressing GFP-FIP3 (A) or GFP-FIP3I738E (B) infected for 16h. FIP3 distribution was examined using anti-GFP antibody followed by protein A-gold particles. Panels a in A and B show gold particles in mammalian cells. Panels c and d in A and B illustrate gold particles localized within the PV on vesicles (arrowheads), and sometimes in the vicinity of the IVN. Some gold particles were also detected on the limiting membrane on internal vesicles (arrows). hc, host cell; P, parasite. All bars, 300 nm.

Figure 8. Abundance of mammalian Rab11A vesicles containing FIP3 in the PV A. Fluorescence microscopy of HeLa cells transfected with GFP-FIP3 and immunostained with anti- Rab11A antibody at interphase (panel a) and during cytokinesis (panel b). The circle highlights the midbody. B. Fluorescence microscopy of HeLa cells transfected with GFP-FIP3 and infected for 24h before immunostaining for Rab11A and TgGRA7. The delocalization of the ERC at the top of the PV or squeezed between two PV is highlighted in panels a and b, respectively. For all images, individual z-slices and orthogonal views are shown. Arrows and squares in panels b-d show intra-PV GFP-FIP3 foci and/or Rab11A foci.

Figure 9. Distribution of mammalian vesicles containing Arf6 or FIP3 in Toxoplasma-infected cells A-B. Fluorescence microscopy of HeLa cells transfected with Arf6-mCherry, uninfected (A) or infected for 24h (B), before immunostaining with anti-TgGRA7 antibody (panel b). For all images, individual z- slices and orthogonal views are shown. Arrows show intra-PV Arf6-mCherry foci (panel a) and arrowheads pinpoint foci associated with the IVN (panel b). C-D. Fluorescence microscopy of HeLa cells co-transfected with Arf6-mCherry and GFP-FIP3, uninfected (C, panel a: at interphase; panel b: end of cytokinesis) or infected for 24h (D). Arrows and squares show intra-PV Arf6-mCherry and/or GFP-FIP3 foci.

Figure 10. Distribution of mammalian vesicles containing Arf6 or FIP3-RBD in Toxoplasma-infected cells A. Quantitative analysis of percent of PV containing Arf6-mCherry or GFP-FIP3, and the percent of Arf6- mCherry foci that colocalized with GFP-FIP3/GFP-FIP3-RBD based on experiments described in Figure 9 and below. B-C. Fluorescence microscopy of HeLa cells co-transfected with Arf6-mCherry and GFP- FIP3I738E, uninfected or infected for 24h, before immunostaining with anti-TgGRA7 antibody. For all images, individual z-slices and orthogonal views are shown. Arrows and squares in panels b-d show intra- PV GFP-FIP3-RBD foci.

Figure 11. Enumeration of foci with Arf6, FIP3 or FIP3-RBD vesicles within the Toxoplasma PV A-B. Quantification of data described in Figures 9 and 10. Intra-PV vesicles were counted in all the PV or all the positive PV for Arf6-mCherry (in A) or GFP-FIP3/GFP-FIP3-RBD (in B). Tables show two-tailed bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

p values compared to control conditions corresponding to mono-transfected HeLa cells with Arf6-mCherry, GFP-FIP3 or GFP-FIP3Δ687-712.

Figure 12. Comparative distribution of mammalian vesicles containing Rab11-FIP3-ABD and Rab11- FIP3-RBD-ABD in the Toxoplasma PV A-B. Fluorescence microscopy of HeLa cells co-transfected with GFP-FIP3Δ687-712 (A) or GFP-FIP3I738EΔ687- 712 (B, panels b and c) and infected for 24h before immunostaining with anti-TgGRA7 antibody. DAPI in blue. For all images, individual z-slices and orthogonal views are shown. Arrows show intra-PV foci. C. Percent of PV with intra-PV foci corresponding to vesicles with GFP-FIP3-ABD or GFP-FIP3-RBD-ABD (panel a) and enumeration of mammalian intra-PV vesicles with GFP-FIP3-ABD or GFP-FIP3-RBD-ABD mutants in all PV (panel b) or PV positive for GFP (panel c ). Two-tailed p-values are shown.

Figure 13. Graphical summary of the differential internalization of mammalian FIP recycling vesicles into the Toxoplasma PV The percentage of positive PV contain FIP-associated vesicles is shown. The percentage of PV containing recycling vesicles marked with a WT or RBD-mutant FIP from Class I or Class II is indicated, revealing selectivity for PV internalization among FIP vesicles. No vesicles with Class I FIP-RBD mutants are detected in the PV while vesicles with class II FIP-RBD mutants are still internalized, with PV contain vesicles with FIP4-RBD. Vesicles with FIP3-RBD or FIP3-ABD are internalized into PV. Very few PV display vesicles with the dual mutant FIP3-RBD-ABD.

Figure 14. Changes in gene expression for host recycling Rab proteins and effectors upon a 24h Toxoplasma infection in fibroblasts

RNA-seq data showing Log2 fold change in gene transcripts identified in the database and padj values of statistical significance.

bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplemental information

Figure S1. Distribution of Rab11A vesicles cells in infected cells with Δgra2Δgra6 mutants Fluorescence microscopy of Rab11A-expressing VERO cells infected with Δgra2Δgra6 mutants for 24h, before immunostaining with anti-TgGRA7 (PVM/IVN) and TgNTPase (PV lumen) antibodies.

Figure S2. Distribution of FIP vesicles cells in infected cells with Δgra2Δgra6 mutants Fluorescence microscopy of HeLa cells transfected with GFP-FIP1C, GFP-FIP2, GFP-FIP5, GFP-FIP3, GFP-FIP3I738E, GFP-FIP4 or GFP-FIP4YMD617-619AAA, infected with Toxoplasma for 24h before immunostaining with anti-TgGRA7 antibody. DAPI in blue. For all images, individual z-slices are shown.

Figure S3. Description of selected Rabs and their effectors genes with differential expression upon Toxoplasma infection

Table SI. List of genes with differential expression upon Toxoplasma infection

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Figure 1 A uninfected αRab11 infected RFP-Tg αRab11 TgGRA7

PV

IVN

B uninfected GFP-Rab11A infected RFP-Tg GFP-Rab11A TgGRA7

PV

C uninfected GFP-Rab11B infected RFP-Tg GFP-Rab11B TgGRA7

PV

infected

uninfected GFP-Rab11A αRab11A Da

8.00µm infected b PV

7.00µm c PV

9.00µm bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2

A Class I FIPs a uninfected infected b uninfected infected

GFP-FIP2 GFP-FIP1C

8.00 µm

PV 8.00 µm PV 8.00 µm

c uninfected infected GFP-FIP5

8.00 µm 8.00 µm PV

B Class II FIPs

a uninfected infected infected GFP-FIP3

PV

PV

uninfected uninfected b (interphase) (cytokinesis) infected

GFP-FIP4

PV bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3 A GFP-FIP1C TgGRA7 Class I FIPs

PV

GFP-FIP2 TgGRA7

PV

GFP-FIP5 TgGRA7

PV

B GFP-FIP3 TgGRA7 Class II FIPs PV

GFP-FIP4 TgGRA7

PV

C FIP Class % PV with GFP-FIP foci (# of PV) WT Δgra2Δgra6 FIP1C I 12% (25) 0% (22) FIP2 I 57% (21) 0% (23) FIP5 I 52% (21) 5% (22) FIP3 II 88% (24) 0% (20) FIP4 II 50% (24) 8% (24) bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4

A Class I FIP-RBD mutants infected a uninfected infected c uninfected

GFP-FIP1CI621E

5.00 µm

PV PV PV 5.00 µm

GFP-FIP5I630E b

GFP-FIP2I481E

B Class II FIP-RBD mutants

a uninfected infected b uninfected infected

8.00 µm 8.00 µm

PV PV 8.00 µm 8.00 µm

GFP-FIP3I738E GFP-FIP4YMD617-619AAA

8.00 µm 8.00 µm bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5 A Class I FIP-RBD mutants GFP-FIP1CI621E TgGRA7 PV

GFP-FIP2I481E TgGRA7

PV

GFP-FIP5I630E TgGRA7

PV

GFP-FIP3I738E TgGRA7 B Class II FIP-RBD PV mutants

GFP-FIP4YMD617-619AAA TgGRA7

PV

C FIP mutant Class % PV with GFP-FIP-RBD foci (# of PV)

FIP1CI621E I 0% (24) FIP2I481E I 0% (19) FIP5I630E I 0% (23) FIP3I738E II 74% (23) FIP4YMD617-619AAA II 100% (23) bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6

25 A Class I FIPs Class II FIPs 20

FIP foci FIP 15 -

10 in PV all 5

0 Number of of GFP Number -5 FIP1C FIP2 FIP5 FIP3 FIP3I738E FIP4 FIP4YMD617-619AAA

FIP1c FIP2 FIP3 FIP3I738E FIP4 FIP4YMD617- 619AAA FIPC1 FIP2 0.0048 FIP3 0.00002 ns FIP3I738E 0.0006 ns 0.026 FIP4 0.0068 ns ns 0.015 FIP4YMD617- 0.0000004 0.0017 0.0001 ns 0.00005 619AAA FIP5 0.0056 ns ns 0.007 ns 0.00001

25 B Class I FIPs Class II FIPs 20 FIP foci foci FIP

- 15

10

in positive PV in positive 5 Number of of GFP Number 0 FIP1C FIP2 FIP5 FIP3 FIP3I738E FIP4 FIP4YMD617-619AAA

FIP1c FIP2 FIP3 FIP3I738E FIP4 FIP4YMD617- 619AAA FIPC1 FIP2 ns FIP3 0.05 ns FIP3I738E 0.0014 ns 0.0068 FIP4 0.024 ns ns 0.036 FIP4YMD617- 0.00002 ns 0.0003 ns 0.02 619AAA FIP5 0.05 ns ns 0.009 ns 0.0007 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 7 A GFP-FIP3 B GFP-FIP3I738E a b a b hc

hc P P

c c hc P

P P PV

PV

c PV

IVN

d PV d PV P IVN

P bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 8

A uninfected αRab11A GFP-FIP3 a

b

TgGRA7 αRab11A GFP-FIP3 B infected a

PV

b TgGRA7 αPVRab11A GFP-FIP3 PV

TgGRA7 αRab11A GFP-FIP3 c i

i ii

PV ii

6.00 µm 6.00 µm

PV TgGRA7 αRab11A GFP-FIP3 d

PV bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A uninfected Arf6-mCherry Figure 9

6.00 µm

infected Arf6-mCherry Ba

PV

TgGRA7 b PV IVN

Arf6-mCherry b C uninfected Arf6-mCherry GFP-FIP3 a

6.00 µm 6.00 µm GFP-FIP3

6.00 µm 6.00 µm

D infected Arf6-mCherry GFP-FIP3

PV bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 10

A %PV positive %PV positive with %Arf6 foci I738E colocalized with with Arf6 foci FIP3/FIP3 foci FIP3/FIP3 I738E foci (PV #) (PV #) (foci #) Arf6 86% (29) FIP3 88% (24) FIP3I738E 74% (23)

Exp. 1 61% (23) 70% (23) 80% (54) Arf6+FIP3 Exp. 1 25% (20) 80% (24) 29% (7) Arf6+FIP3 I738E Exp. 2 61% (23) 87% (23) 65% (77) Arf6+FIP3 Exp. 2 21% (19) 68% (19) 0% (11) Arf6+FIP3 I738E

Arf6-mCherry Arf6-mCherry B uninfected GFP-FIP3I738E infected GFP-FIP3I738E TgGRA7 PV

Cinfected Arf6-mCherry GFP-FIP3I738E TgGRA7

PV bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A Figure 11 15 15

10 10

5

in positive PV 5 0 Number of Arf6 foci Arf6 of Number

Number of Afr6 foci Afr6 in of all Number PV -5 0 control FIP3 FIP3 I738E FIP3 FIP3 I738E control FIP3 FIP3I738E FIP3 FIP3I738E (monotransf.) (monotransf.)

All PV with Positive PV Arf6 foci with Arf6 foci Exp. 1 0.586 0.429 Arf6+FIP3 Exp. 1 0.000089 0.0058 Arf6+FIP3 I738E Exp. 2 0.540 0.0457 Arf6+FIP3 Exp. 2 0.00036 0.644 Arf6+FIP3 I738E B 20 20

15 15

10 10 5 in positive PV

5

0 FIP3 foci of Number

Number of FIP3 foci FIP3 foci of inNumber all PV -5 0

control FIP3 FIP3 control FIP3 FIP3 (Exp. 1) (Exp. 2) (Exp. 1) (Exp. 2) 25 (monotransf .) 25 (monotransf.)

20 20

15 15 10 10

5

in positive PV 5 0 Number of FIP3mut FIP3mutfoci of Number Number of FIP3mut FIP3mutfoci of inNumber all PV -5 0 control FIP3I738E FIP3I738E control FIP3I738E FIP3I738E (monotransf .) (Exp. 1) (Exp. 2) (monotransf.) (Exp. 1) (Exp. 2)

All PV with Positive PV FIP3 foci with FIP or FIP3I738E foci Exp. 1 0.156 0.0147 FIP3 Exp. 2 0.0997 0.0403 FIP3I738E Exp. 2 0.0248 0.0156 FIP3 Exp. 2 0.483 0.433 FIP3I738E bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 12

A GFP-FIP3Δ687-712 TgGRA7 PV

B a GFP-FIP3I738EΔ687-712 TgGRA7 PV

b GFP-FIP3I738EΔ687-712

PV

C

All Foci in Positive PVs b 20 0.0014 a c 20 0.0147 155 FIP mutants % PV with FIP3mut 15 foci (# PV) 10 10 FIP3Δ687-712 95% (20) 5

foci in all all PV in foci FIP3I738EΔ687-712 37% (27) 5

Number of FIP3mut of Number 0 focipositive in PV Number of FIP3mut of Number -55 0

FIP3Δ687-712FIP3 I738EΔ687-712 FIP3Δ687-712 FIP3I738EΔ687-712

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Figure 13

FIP1C Toxoplasma PV (%) I621E I621E

FIP1C Class I Class II FIP3

FIP1C Arf6 712 687 - FIP1C 88% Δ 712 FIP3 FIP 687 -

12% Δ I630E I630E

I738E %

95 I738E FIP2 FIP2

FIP3 Arf6 FIP3 FIP2 37% 74% 57% FIP4 FIP5 Arf6 619AAA FIP5 FIP5 I4815 I481E 50% - YMD617

FIP5 52% Arf6 FIP4 100% bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 14

RECYCLING Rab & EFFECTORS Log2-FC Padj value Rab Rab5B - 1.39 4.7E-10 Rab15 +2.24 3.3E-19 Rab GAP TBC1D4 +1.63 9.2E-15 TBC1D11 (RABGAP1) - 1.51 5.8E-10 EVI5-L - 1.03 8.2E-05 Rab GEF Rabin-8 (RAB3IP) +1.97 0.00155 GRAB (RAB31L1) - 0.63 0.04644 Mss4 (RABIF) +1.02 0.00174 FERARI components (Rab11) FIP5 - 0.59 0.01606 Rabenosyn-5 - 0.74 0.00845 VPS45 - 1.02 0.00004 VIPAS39 - 1.12 0.00387 EHD1 +1.38 3.1E-08 SNX1 - 0.82 0.00038 Proteins with TRAPP complexes Kalirin (KALRN) - 0.84 0.04571 TRAPPC3L +2.75 1.1E-06 TRAPPC6A - 2.07 0.00229 TRAPPC1 - 1.33 7.2E-07 TRAPPC9 - 1.04 0.00121 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure S1

Δgra2Δgra6 GFP-Rab11A TgGRA7 TgNTPase z1

z2

PV bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure S2

Class I FIPs

GFP-FIP1C GFP-FIP2 GFP-FIP5

Class II FIPs

GFP-FIP3 GFP-FIP3I738E GFP-FIP4 GFP-FIP4YMD617-619AAA bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure S3

Selected Rab Functions/Localization References and effectors

Rab5b Localizes to a TfR-positive endosomal compartment Wilson and Wilson, 1992; Bucci et al., 1995

Rab8 Regulates the exocytic trafficking of proteins from the TGN and RE Hattula et al., 2006; containing TfR to the plasma membrane, Peranen, 2011 and it co-distributes with Arf6 on tubular organelles.

Rab15 Co-localizes with Rab4, Rab5, and the TfR on SE and with Rab11 on the ERC. Zuk and Elferink, 1999; 2000 Regulates the TfR trafficking through SE and the ERC, thus intracellular iron levels.

Rab35 Major player in the fast recycling pathway and cytokinesis Klinkert and Echard, 2016 localized at the plasma membrane and endosomes.

Rab36 Functions/Localization Kobayashi et al., 2014

TBC1D4 Has several domains and phosphorylation sites. Kane et al., 2002; Involved in the shuttle of the glucose transporter 4 (GLUT4) Mîinea et al., 2005; between GLUT4 storage vesicle (GSV) and the plasma membrane Sano et al., 2003; Acts through the phosphorylation of S/T residues by upstream kinases, Treebak et al., 2010 resulting in inactivation of the enzymatic function of RabGAP and the translocation of GSV to the plasma membrane to expose GLUT4 and enhance glucose uptake in cells.

TBC1D11 GAP that binds Rab36 and co-localizes with RE containing TfR. Kanno et al., 2010

EV15-L GAP for Rab11 and is a modulator of cell cycle progression, Lim and Tang, 2013; cytokinesis and cellular membrane traffic. Kanno et al., 2010 Co-localizes with RE containing TfR.

VIPAS39 Involved in the sorting of lysosomal proteins. van der Kant et al., 2015

Rabenosyn-5 Stimulates TfR recycling, activating the fast pathway Mohrmann et al., 2002; and switching off the slow recycling route through Rab11-positive membranes. Nielsen et al., 2000 Associated with early endosomes containing both Rab5 and Rab4; Rab4 regulates recycling vesicle formation Rabenosyn-5 binds to VPS45

VPS45 Forms a complex with Rabenosyn-5 that provides a regulatory function Olson et al., 1997; for the pairing of SNARE after Rab5-Rab4 membrane tethering of during recycling from early Prekeris et al., 1998; endosomes to the plasma membrane. Rahajeng et al., 2010 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446625; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Name Notes Gene ID HFF RH 1 HFF RH 2 HFF 1 HFF 2 log2FoldChangepvalue padj GeneName Chrom Strand Start End GeneLength GeneType GeneDescription HFF RH 1 r HFF RH 2 reHFF 1 readc HFF 2 readc HFF RH 1 f HFF RH 2 fpHFF 1 fpkm HFF_2_fpkm

Rab4 No change Rab5A No change Rab5B ENSG000001 811.8378489 903.074121 2346.15565 2238.19015 -1.3916 1.62E-11 4.71E-10 RAB5B chr12 + 55973913 55996683 6588 protein_coding RAB5B member RAS oncogene family 522 782 3352 2868 9.30587363 9.78169537 25.1379941 21.651144 Rab8A No change Rab10 No change Rab11A No change Rab11B No change Rab13 No change Rab14 No change Rab15 ENSG000001 1087.1162 937.718908 198.07937 214.610283 2.238 3.73E-21 3.28E-19 RAB15 chr14 - 64945814 64972776 4379 protein_coding RAB15 member RAS oncogene family 699 812 283 275 18.7474614 15.280658 3.19294686 3.12329525 Rab22A No change Rab35 No change Rab36 No change TBC1D1 GAP; no change TBC1D3 GAP; no change TBC1D4 GAP ENSG000001 1158.657466 1069.3691 363.962093 337.913645 1.6299 2.47E-13 9.15E-12 TBC1D4 chr13 - 75284665 75482114 6658 protein_coding TBC1 domain family member 4 745 926 520 433 13.1417354 11.4611492 3.85868879 3.23444222 TBC1D10A GAP; no change TBC1D10B GAP; no change TBC1D10C GAP; no change TBC1D11 GAP; alias RABGAP1 ENSG000000 558.3329267 595.890341 1520.94159 1847.98964 -1.5105 2.05E-11 5.80E-10 RABGAP1 chr9 + 122940833 123104866 9162 protein_coding RAB_GTPase_activating_protein_1 359 516 2173 2368 4.60197653 4.64109422 11.717896 12.8542488 TBC1D15 GAP; no change TBC1D17 GAP; no change TBC1D18 GAP; no change TBC1D25 GAP; no change TBC1D30 GAP; no change EVI5 GAP; no change EVI5-L GAP ENSG000001 382.5902506 412.272968 779.718792 876.390355 -1.033 8.72E-06 8.21E-05 EVI5L chr19 + 7830233 7864976 6252 protein_coding ecotropic viral integration site 5-like 246 357 1114 1123 4.62121714 4.70554811 8.80331866 8.93338558 RUTBC2 GAP; no change RUTBC3 GAP; no change USP6NL GAP; no change Rabex-5 GEF; no change GEF; Alias - RAB3IP, RAB3A Interacting Protein, Rabin-3; GEF which may activate RAB8A and 8B; promotes exchange of GDP to GTP, mediates release of GDP from Rabin-8 RAB8A and 8B but not from RAB3A or RAB5 ENSG000001 74.65175622 48.5027022 14.6984691 10.1452134 1.9676 0.00024391 0.0015486 RAB3IP chr12 + 69738681 69823204 12612 protein_coding RAB3A_interacting_protein 48 42 21 13 0.4469897 0.27442666 0.08226508 0.05126426 RAB3A_interacting_protein_(rabin3)- GRAB GEF for Rab3A; alias RAB3IL1 ENSG000001 315.714719 263.300383 466.15145 441.706982 -0.63273 0.01371 0.046437 RAB3IL1 chr11 - 61897301 61920269 3006 protein_coding like 1 203 228 666 566 7.93135337 6.25038604 10.9462523 9.36446018 GEF; Alias (RABIF = RAB interacting factor); Stimulates GDP release from both YPT1, RAB3A and RAB10 but is Mss4 less active on these proteins than on SEC4 ENSG000001 628.3189482 437.679146 267.372153 244.265522 1.0197 0.00027975 0.0017412 RABIF chr1 - 202878957 202889135 2430 protein_coding RAB_interacting_factor 404 379 382 313 19.5260919 12.8526863 7.76671359 6.40609473 C9Orf72 GEF; no change; alias DENND9, DENNL72 REI-1 GEF; no change DennD1A GEF; no change DennD1B GEF; no change DennD1C GEF; no change DennD4a GEF; no change DennD4c GEF; no change alias - RAB10GEF DennD6 GEF; no change

Rab11FIP5 Alias - RIP11, GAF1, Gamma-SNAP-Associated Factor 1; ENSG000001 1367.060286 1523.21581 2127.77839 2261.60218 -0.59139 0.0037719 0.016055 RAB11FIP5 chr2 - 73073382 73156721 5932 protein_coding RAB11 family interacting protein 5 (clas 879 1319 3040 2898 17.4031543 18.3233393 25.3193552 24.2969931

ARF6 No change RAB4 No change RAB8 No change RAB10 No change RAB11 No change RAB22A No change RAB35 Rabenosyn5 ENSG000001 399.6979448 467.704628 768.519958 702.360926 -0.74164 0.0017607 0.0084479 RBSN chr3 - 15070073 15099163 7921 protein_coding rabenosyn_RAB_effector 257 405 1098 900 3.81059987 4.21343192 6.84861145 5.65090217 RAB11FIP2 No change RAB11FIP3 (NUF) No change RAB11FIP5 (RIP11) ENSG000001 1367.060286 1523.21581 2127.77839 2261.60218 -0.59139 0.0037719 0.016055 RAB11FIP5 chr2 - 73073382 73156721 5932 protein_coding RAB11 family interacting protein 5 (clas 879 1319 3040 2898 17.4031543 18.3233393 25.3193552 24.2969931 Phospholipase D2 No change PtdIns(4)P5K No change SEC10 (EXOC5) No change JIP3 No change JIP4 No change RME-4 (connecdenn) No change TBC1D10C (EP164C) No change ASAP1 No change ACAP1 No change Loner (Schizo/ARFGEP100No change

Proteins associated with TRAPP complexes Alias - Kalirin RhoGEF Kinase, KALRN, ARHGEF24, HAPIP; GEF of Rho GTPase family members; kalirin and TRAPP may form a dual GEF complex to choreograph Rab11 and Kalirin Rac1 at RE ENSG000001 244.1734526 225.191117 299.5688 570.473152 -0.84382 0.013458 0.045707 KALRN chr3 + 124080023 124726325 24490 protein_coding kalirin_RhoGEF_kinase 157 195 428 731 0.75292394 0.65615554 0.86344601 1.48451259 TRAPPC3L ENSG000001 88.64896051 75.0637057 11.1988336 7.02360926 2.7477 7.36E-08 1.11E-06 TRAPPC3L chr6 - 116494989 116545610 2757 protein_coding trafficking protein particle complex 3-lik 57 65 16 9 2.42816574 1.94284235 0.28672358 0.16235327 TRAPPC6A ENSG000000 7.776224606 13.8579149 41.295699 85.0637121 -2.0685 0.00038322 0.0022905 TRAPPC6A chr19 - 45162928 45178237 933 protein_coding trafficking_protein_particle_complex_6A 5 12 59 109 0.6294027 1.05988946 3.12428446 5.81032127 TRAPPC1 ENSG000001 270.6126163 244.823163 642.53308 682.8509 -1.329 4.58E-08 7.24E-07 TRAPPC1 chr17 - 7930345 7932123 1171 protein_coding trafficking_protein_particle_complex_1 174 212 918 875 17.4514932 14.9190077 38.731649 37.1626308 TRAPPC9 ENSG000001 169.5216964 219.416986 396.858667 424.538159 -1.0375 0.00018483 0.0012168 TRAPPC9 chr8 - 139730343 140458579 7055 protein_coding trafficking_protein_particle_complex_9 109 190 567 544 1.81455325 2.21930787 3.97069237 3.83492764

FERARI complex components (Rab11) Rab11FIP5 ENSG000001 1367.060286 1523.21581 2127.77839 2261.60218 -0.59139 0.0037719 0.016055 RAB11FIP5 chr2 - 73073382 73156721 5932 protein_coding RAB11 family interacting protein 5 (clas 879 1319 3040 2898 17.4031543 18.3233393 25.3193552 24.2969931 rabenosyn-5/RABS5 ENSG000001 399.6979448 467.704628 768.519958 702.360926 -0.74164 0.0017607 0.0084479 RBSN chr3 - 15070073 15099163 7921 protein_coding rabenosyn_RAB_effector 257 405 1098 900 3.81059987 4.21343192 6.84861145 5.65090217 VPS45 ENSG000001 264.3916366 324.506174 613.836069 606.371599 -1.0194 4.60E-05 0.00035924 VPS45 chr1 + 150067293 150145327 6262 protein_coding vacuolar protein sorting 45 homolog (S 170 281 877 777 3.1884242 3.69789204 6.91937282 6.17110943 VIPAS39 ENSG000001 101.0909199 161.675674 273.671497 323.086026 -1.1177 0.00071159 0.0038735 VIPAS39 chr14 - 77426675 77457952 3695 protein_coding VPS33B interacting protein apical-basola 65 140 391 414 2.06604203 3.12229953 5.2280835 5.57238598 EHD1 ENSG000001 6623.78812 5363.01307 1988.4929 2508.98931 1.382 1.46E-09 3.05E-08 EHD1 chr11 - 64851642 64888296 7101 protein_coding EH_domain_containing_1 4259 4644 2841 3215 70.4414627 53.8931625 19.7665976 22.517323 Possible FERARI interactors SNX1 ENSG000000 1032.682628 1110.94284 1937.39822 1900.2765 -0.82473 4.87E-05 0.00037702 SNX1 chr15 + 64094123 64146090 9780 protein_coding sorting_nexin_1 664 962 2768 2435 7.97387572 8.10582434 13.9832278 12.3827015 SYX6 no change SYX7 no change

Other Rab and RAB associated proteins with expression changes in 24h Toxoplasma RH infection of HFF Rab33A ENSG000001 31.10489843 57.7413121 3.49963551 11.7060154 1.9859 0.0017966 0.0085844 RAB33A chrX + 130171649 130184870 1258 protein_coding RAB33A_member_RAS_oncogene_family 20 50 5 15 1.86719464 3.27529434 0.19636748 0.59301532 Rab38 ENSG000001 172.6321863 101.624709 65.7931476 27.314036 1.3838 0.0035627 0.015331 RAB38 chr11 - 88113242 88175467 1465 protein_coding RAB38 member RAS oncogene family 111 88 94 35 8.89868005 4.95000934 3.17008152 1.18818951 Rab27B ENSG000000 150.8587574 92.3860993 65.0932205 22.6316298 1.2853 0.010322 0.036821 RAB27B chr18 + 54717860 54895516 7432 protein_coding RAB27B_member_RAS_oncogene_family 97 80 93 29 1.53287334 0.88704419 0.6182405 0.19406517 Rab6B ENSG000001 130.6405734 88.9216206 55.9941682 43.7024576 1.043 0.011538 0.040293 RAB6B chr3 - 133824239 133895836 6005 protein_coding RAB6B_member_RAS_oncogene_family 84 77 80 56 1.64288253 1.05666831 0.65819894 0.46379953 Rab27A ENSG000000 612.766499 718.301922 340.164572 312.940812 1.0017 3.60E-05 0.00029003 RAB27A chr15 - 55202966 55319113 5605 protein_coding RAB27A_member_RAS_oncogene_family 394 622 486 401 8.25583193 9.14483215 4.28391508 3.55814832 Rab7B ENSG000002 45.10210272 46.1930497 424.855751 362.886478 -2.9638 2.63E-20 2.08E-18 RAB7B chr1 - 205976740 206003461 3686 protein_coding RAB7B_member_RAS_oncogene_family 29 40 607 465 0.92402326 0.89426376 8.13604911 6.27412135 Rab30 ENSG000001 209.9580644 203.249419 503.947514 418.294951 -1.123 2.51E-05 0.00021154 RAB30 chr11 - 82973133 83071923 11832 protein_coding RAB30_member_RAS_oncogene_family 135 176 720 536 1.34003409 1.22578832 3.00645381 2.25300378 Rab3B ENSG000001 3295.563988 3534.92313 6194.35486 6283.00868 -0.85547 7.01E-06 6.77E-05 RAB3B chr1 - 51907956 51990764 12844 protein_coding RAB3B_member_RAS_oncogene_family 2119 3061 8850 8051 19.3763021 19.6392095 34.0426355 31.1748823 Rab31 ENSG000001 931.5917078 1218.34169 1790.41353 1975.195 -0.78841 0.00065687 0.0036158 RAB31 chr18 + 9708165 9862551 5586 protein_coding RAB31_member_RAS_oncogene_family 599 1055 2558 2531 12.5940708 15.5636874 22.6245427 22.5344263 Rab1A ENSG000001 1877.18062 1912.39226 2962.0915 3049.02682 -0.6547 0.00072965 0.0039545 RAB1A chr2 - 65070701 65130106 2951 protein_coding RAB1A_member_RAS_oncogene_family 1207 1656 4232 3907 48.0372679 46.2436488 70.8527401 65.8460146 Rab18 ENSG000000 1141.549772 1267.99921 1614.0319 2008.75225 -0.57484 0.010418 0.037054 RAB18 chr10 + 27504174 27542237 6223 protein_coding RAB18_member_RAS_oncogene_family 734 1098 2306 2574 13.852766 14.53997 18.3079494 20.5714088

GAP for Rab7A; signal effector linking RAC1 and Rab7A TBC1D2 leading to Rab7A inactivation ENSG000000 978.2490555 780.662539 1950.69683 1314.97573 -0.86568 0.0012333 0.0062215 TBC1D2 chr9 - 98198999 98255721 3681 protein_coding TBC1_domain_family_member_2 629 676 2787 1685 20.0689692 15.133586 37.4068684 22.7661388 TBC1D19 possible GAP ENSG000001 155.5244921 140.888801 411.557136 483.848638 -1.5433 2.19E-08 3.71E-07 TBC1D19 chr4 + 26576437 26755351 3434 protein_coding TBC1 domain family member 19 100 122 588 620 3.420109 2.92765913 8.45974429 8.97938703 Rab11A binding protein that plays a role in neurite TBC1D12 outgrowth ENSG000001 143.0825328 181.30772 255.473392 295.77199 -0.73026 0.014657 0.049015 TBC1D12 chr10 + 94402504 94535930 6921 protein_coding TBC1 domain family member 12 92 157 365 379 1.56120242 1.86935496 2.60557883 2.72348917 TBC1D23 putative GAP ENSG000000 614.3217439 724.076054 1110.78431 1140.9463 -0.73303 0.00097392 0.0050768 TBC1D23 chr3 + 100261000 100325251 5168 protein_coding TBC1_domain_family_member_23 395 627 1587 1462 8.97666109 9.99783596 15.1717131 14.0695485 putative GAP for RAB7A; can displace Rab7A and retromer CSC subcomplex from the endosomal membrane to cytosol; required for retrograde transport TBC1D5 of cargo proteins from endosomes to TGN ENSG000001 788.5091751 794.520454 1461.44779 1164.35833 -0.71361 0.0017962 0.0085844 TBC1D5 chr3 - 17157162 18444817 11269 protein_coding TBC1_domain_family_member_5 507 688 2088 1492 5.28400012 5.03111252 9.1543037 6.5847416 TBC1D2B putative GAP ENSG000001 597.2140498 787.591497 1037.99189 1092.56144 -0.60408 0.011636 0.040556 TBC1D2B chr15 - 77984036 78077724 9763 protein_coding TBC1_domain_family_member_2B 384 682 1483 1400 4.61942769 5.75654702 7.50478168 7.1318145 TBC1D8B putative RAB11B GAP ENSG000001 133.7510632 183.617372 314.267269 343.376453 -1.0041 0.00075485 0.0040744 TBC1D8B chrX + 106802680 106876145 9503 protein_coding TBC1_domain_family_member_8B 86 159 449 440 1.06286464 1.37878759 2.33434914 2.30275238

DENND3 GEF for Rab12; paralog of DENND4A ENSG000001 553.667192 416.892273 685.228633 1235.37483 -0.94137 0.0030951 0.013596 DENND3 chr8 + 141117278 141195808 14670 protein_coding DENN/MADD domain containing 3 356 361 979 1583 2.85010016 2.02786042 3.29710501 5.36668486

Rab escort protein; substrate-binding subunit (component A) of the Rab geranylgeranyltransferase choroideremia- CHML (GGTase) complex ENSG000002 1241.085447 1145.58763 421.356116 518.186283 1.3136 1.44E-08 2.50E-07 CHML chr1 - 241628853 241635930 7078 protein_coding like (Rab escort protein 2) 798 992 602 664 13.2413593 11.5494708 4.20209759 4.66565706 catalyzes transfer of geranylgeranyl to both cysteines of RABGGTB Rab proteins with C-term XXCC, CCXX, and XCXC motifs ENSG000001 797.8406446 725.23088 423.455897 475.264226 0.7429 0.0013253 0.006617 RABGGTB chr1 + 75786197 75795079 5913 protein_coding Rab geranylgeranyltransferase beta sub 513 628 605 609 10.1894261 8.75210936 5.05507608 5.12229641 Rab effector protein acting as linker between gamma- adaptin, RAB4A and RAB5A; stimulates RABGEF1 RABEP1 mediated nucleotide exchange on RAB5A ENSG000000 1281.521815 1444.68763 875.608805 955.210859 0.56362 0.0085394 0.031495 RABEP1 chr17 + 5282265 5385812 6531 protein_coding rabaptin RAB GTPase binding effector 824 1251 1251 1224 14.8179378 15.7847823 9.46363162 9.32088235 binds to both GTP and GDP; may play a role in cell RABL6 growth and survival; alias - RBEL1; PARF; C9orf86 ENSG000001 1755.871516 1727.62006 1157.67943 1236.93563 0.53119 0.0085689 0.031567 RABL6 chr9 + 136807943 136957733 10193 protein_coding RAB member RAS oncogene family-like 1129 1496 1654 1585 13.0086478 12.094573 8.0170332 7.73361476 Rab gene family; plays an essential role in male fertility; binds, in a GFP-regulated manner, to a specific set of effector proteins including key proteins involved in cilia RABL2A development and function ENSG000001 23.32867382 48.5027022 121.087389 110.816946 -1.5129 0.00072858 0.0039513 RABL2A chr2 + 113627229 113643396 6008 protein_coding RAB member of RAS oncogene family-l 15 42 173 142 0.29322539 0.57607674 1.42264447 1.17547585

RIN3 Alias Ras and Rab interactor 3; GEF for RAB5B and RAB31 ENSG000001 304.8280046 497.73011 1184.97658 996.572113 -1.3865 1.37E-06 1.57E-05 RIN3 chr14 + 92513774 92688994 8683 protein_coding Ras_and_Rab_interactor_3 196 431 1693 1277 2.65110243 4.09042506 9.6331286 7.31436096 GEF that activates RAB6A; may be required for efficient fusion of endosome-derived vesicles with the Golgi compartment; RIC1-RGP1 complex participates in RIC1 recycling of mannose-6-phosphate receptors ENSG000001 813.3930938 1057.82084 1229.77192 2352.1287 -0.89393 0.0048029 0.019579 RIC1 chr9 + 5629025 5776557 8591 protein_coding RIC1 homolog RAB6A GEF complex par 523 916 1757 3014 7.14987103 8.7864355 10.1043468 17.4483684 RGP1 see above ENSG000001 357.7063319 354.531656 590.738474 688.313707 -0.82301 0.00065496 0.0036093 RGP1 chr9 + 35749287 35758575 7116 protein_coding RGP1 homolog RAB6A GEF complex pa 230 307 844 882 3.79605184 3.55519484 5.85985282 6.16435921

Alias - Rabenosyn5, ZFYVE20; Rab4/Rab5 effector protein acting in EE membrane fusion and membrane trafficking of recycling endosomes; FYVE zinc finger family of RBSN proteins ENSG000001 399.6979448 467.704628 768.519958 702.360926 -0.74164 0.0017607 0.0084479 RBSN chr3 - 15070073 15099163 7921 protein_coding rabenosyn_RAB_effector 257 405 1098 900 3.81059987 4.21343192 6.84861145 5.65090217

Alias - RASSF4, Ras and Rab Interactor 2, MACS; preferentially binds GTP-bound RAB5; may function as GEF of RAB5B; may function as an upstream activator RIN2 and/or downstream effector for RAB5B ENSG000001 595.6588048 630.535128 907.805452 1093.34184 -0.68972 0.0026815 0.012054 RIN2 chr20 + 19886521 20002457 5053 protein_coding Ras_and_Rab_interactor_2 383 546 1297 1401 8.90204353 8.90439291 12.6815074 13.7893606

Search with ARF ASAP3 ENSG000000 272.1678612 260.990731 757.321125 765.573409 -1.4784 3.24E-10 7.71E-09 ASAP3 chr1 - 23428563 23484568 4988 protein_coding ArfGAP with SH3 domain ankyrin repea 175 226 1082 981 4.12051825 3.73373048 10.7171921 9.78132873 ASAP2 ENSG000001 1048.235077 1084.38184 598.437672 740.600576 0.65652 0.0042513 0.01772 ASAP2 chr2 + 9206765 9405683 7248 protein_coding ArfGAP with SH3 domain ankyrin repea 674 939 855 949 10.9214914 10.6759954 5.82811525 6.51183399 ACAP2 ENSG000001 1780.755435 2046.3521 1338.26062 1321.99934 0.51539 0.012864 0.04408 ACAP2 chr3 - 195274736 195443078 11139 protein_coding ArfGAP with coiled-coil ankyrin repeat 1145 1772 1912 1694 12.0725641 13.1092693 8.48050833 7.56349448 AGAP1 ENSG000001 790.06442 741.398447 349.263624 383.957306 1.0378 6.84E-06 6.63E-05 AGAP1 chr2 + 235494089 236131800 13910 protein_coding ArfGAP with GTPase domain ankyrin re 508 642 499 492 4.28920517 3.80337256 1.77236684 1.75911108 ARFGEF3 ENSG000001 603.4350295 539.303855 243.574632 349.619661 0.91401 0.00098619 0.0051374 ARFGEF3 chr6 + 138161921 138344663 14877 protein_coding ARFGEF_family_member_3 388 467 348 448 3.06306774 2.58679783 1.15569726 1.49767625 SMAP2 ENSG000000 785.3986852 935.409256 588.638693 426.879363 0.73844 0.0046841 0.019182 SMAP2 chr1 + 40344850 40423326 3631 protein_coding small_ArfGAP2 505 810 841 547 16.334482 18.383142 11.4432648 7.49232089 ARFGAP2 ENSG000001 567.6643963 553.16177 886.807639 873.268751 -0.63787 0.0034984 0.015106 ARFGAP2 chr11 - 47164299 47177125 5918 protein_coding ADP ribosylation factor GTPase activatin 365 479 1267 1119 7.24366141 6.6699338 10.5774713 9.40395231 ARAP1 ENSG000001 701.4154595 759.875667 1133.88191 1076.17302 -0.58432 0.0064717 0.025128 ARAP1 chr11 - 72685069 72793599 9056 protein_coding ArfGAP with RhoGAP domain ankyrin r 451 658 1620 1379 5.84898309 5.98756789 8.83809777 7.57326484 ARFGAP1 ENSG000001 1037.348362 1173.30346 1569.23656 1666.9366 -0.53855 0.010346 0.036848 ARFGAP1 chr20 + 63272785 63289793 6613 protein_coding ADP ribosylation factor GTPase activatin 667 1016 2242 2136 11.845886 12.6606545 16.7500955 16.0641599