This Is the End: Regulation of Rab7 Nucleotide Binding in Endolysosomal Trafficking and Autophagy

Rab7 – or in yeast, Ypt7p – governs membrane trafficking in the late endocytic and autophagic pathways. Rab7 also regulates mitochondrion-lysosome contacts, the sites of mitochondrial fission. Like all Rab GTPases, Rab7 cycles between an “active” GTP-bound form that binds downstream effectors – e.g., the HOPS and retromer complexes and the dynactin-binding Rab-interacting lysosomal protein (RILP) – and an “inactive” GDP-bound form that cannot bind effectors. Accessory proteins regulate the nucleotide binding state of Rab7: guanine nucleotide exchange factors (GEFs) stimulate exchange of bound GDP for GTP, resulting in Rab7 activation, whereas GTPase activating proteins (GAPs) boost Rab7’s GTP hydrolysis activity, thereby inactivating Rab7. This review will discuss the GEF and GAPs that control Rab7 nucleotide binding, and thus regulate Rab7’s activity in endolysosomal trafficking and autophagy. It will also consider how bacterial pathogens manipulate Rab7 nucleotide binding to support intracellular invasion and immune evasion.

Nucleotide binding regulates Rab-effector interactions (Figure 1; Walworth et al., 1989;Grosshans et al., 2006). Effectors bind specifically to GTP-bound Rab proteins, and therefore the GTP-bound form can be considered the "active" state (Grosshans et al., 2006). GDP-bound Rabs do not bind effectors and can be regarded as "inactive" (Grosshans et al., 2006). Thus, to understand how Rab GTPases function in their cellular context, we must understand how the cell modulates Rab-nucleotide binding.
Accessory proteins control Rab GTPase nucleotide binding (Figure 1; Barr and Lambright, 2010). Guanine nucleotide exchange factors (GEFs) activate Rabs by stimulating exchange of bound GDP for GTP (Ishida et al., 2016;Muller and Goody, 2018). GTPase activating proteins (GAPs) inactivate Rab proteins by boosting their activity for GTP hydrolysis (Fukuda, 2011). This review will discuss how nucleotide binding by Rab7 and its yeast homolog, Ypt7p, is regulated. Rab7 and Ypt7p govern membrane trafficking in the late endocytic and autophagic pathways (Wichmann et al., 1992;Haas et al., 1995;Kim et al., 1999;Guerra and Bucci, 2016). The specific processes controlled by Rab7 and Ypt7p are diverse, as reflected by the large and diverse set of Rab7/Ypt7p effectors.
Rab7 also regulates organelle positioning via its effectors Rab7interacting lysosomal protein (RILP) (Cantalupo et al., 2001;Jordens et al., 2001) and FYVE and coiled-coil containing protein (FYCO1) (Pankiv et al., 2010). RILP recruits the dynein-dynactin motor complex to membranes by binding the p150 Glued protein, a subunit of dynactin (Jordens et al., 2001;Johansson et al., 2007). Rab7 thereby promotes minus end-directed transport of late endosomes and lysosomes on microtubules, that is, transport away from the cell periphery and toward the nucleus (Jordens et al., 2001;Johansson et al., 2007). FYCO1 binds the autophagosomal proteins LC3A and LC3B (Olsvik et al., 2015) and is required for plus end-directed transport, i.e., toward the cell periphery, of autophagosomes on microtubules (Pankiv et al., 2010). FYCO1 also is found on late endosomes and lysosomes, and regulates plus end-directed transport of these organelles as well (Pankiv et al., 2010). The precise mechanism by which FYCO1 regulates organelle positioning remains unknown, but it has been proposed to interact directly or indirectly with kinesin (Pankiv et al., 2010).
Rab7 effectors also regulate protein and lipid kinases that are important for autophagy and for endolysosomal trafficking. Ivy1p, the yeast homolog of the metazoan missing in metastasis (MIM) protein, is a Ypt7p effector (Lazar et al., 2002) that also interacts with the Ragulator complex (Numrich et al., 2015), which in turn regulates the target of rapamycin complex 1 (TORC1) in response to cellular amino acid levels (Jewell et al., 2013). Ivy1p promotes TORC1 activity (Numrich et al., 2015), suggesting that Ivy1p plays a role in signal transduction in response to metabolic state (Jewell et al., 2013). Rubicon, a subunit of the endosomal Vps34/class III phosphatidylinositol 3kinase (PI3KC3) complex, is also a Rab7 effector (Sun et al., 2010). Rubicon negatively regulates Vps34 PI-3-kinase activity (Zhong et al., 2009) and opposes maturation of both autophagosomes (Matsunaga et al., 2009) and endosomes (Sun et al., 2010).
Finally, Rab7 and Ypt7p mediate membrane tethering and fusion. In yeast, the Ypt7p effector responsible for these activities is the homotypic fusion and protein sorting (HOPS)/vacuole protein sorting class C (Vps class C) complex, usually referred to as HOPS (Seals et al., 2000;Wurmser et al., 2000). HOPS is required for membrane tethering (Stroupe et al., 2006) and fusion of lysosomes/vacuoles with late endosomes and autophagosomes (Rieder and Emr, 1997;Jiang et al., 2014;Takats et al., 2014). In yeast, HOPS also is needed for homotypic vacuole fusion (Seals et al., 2000). There is little evidence that HOPS binds Rab7 in metazoans, where HOPS-membrane recruitment appears to be mediated by Arl8b, an Arf-like GTPase (Garg et al., 2011;Khatter et al., 2015). Instead, human Rab7 directly tethers membranes via formation of a Rab7-Rab7 homodimer (Tamura and Mima, 2014).
The following discussion will cover three topics: first, the GEF that activate Rab7/Ypt7p; second, the GAPs that inactivate Rab7/Ypt7p; and third, bacterial effectors, or virulence factors, that modulate Rab7 nucleotide binding to promote intracellular survival and pathogenesis.
Thus, loss of Mg 2+ is proposed to reduce Rab7's affinity for GDP (Kiontke et al., 2017). Second, binding of switch I to Mon1-Ccz1 moves a phenylalanine sidechain in Rab7 away from the area where the guanine ring is found in Rab7-GDP (Figure 3, residue F33). This phenylalanine makes a stabilizing edge-face aromatic interaction with the guanine in Rab7-GDP (Rak et al., 2004). Thus, loss of this interaction should also reduce Rab7 affinity for GDP.
The mechanism of action of Mon1-Ccz1 is quite different from that of other Rab GEFs (Ishida et al., 2016;Muller and Goody, 2018). Mon1-Ccz1 does not belong to the largest and most wellknown class of Rab GEFs, the differentially expressed in normal and neoplastic cells (DENN) domain proteins (Ishida et al., 2016). Rather, the core element in both Mon1 and Ccz1 is a "longin" domain , a small α-β-α sandwich often found in proteins that regulate intracellular traffic (De Franceschi et al., 2014). Longin domains are versatile: some longin domains mediate nucleotide exchange on Rab GTPases, for example as part of the TRAPP complexes (Kim et al., 2006;Cai et al., 2008), whereas others are found in SNARE proteins and appear to have no GEF activity (Rossi et al., 2004).
Recruitment of a Rab5 GAP by Rab7 could, in principle, also promote Rab conversion. Such a feedback loop has been observed in the secretory pathway in yeast. Here, the Golgilocalized Rab, Ypt32p, binds a GAP for an ER-resident Rab, Ypt1p (Rivera-Molina and Novick, 2009). Membrane recruitment of this GAP promotes conversion of Ypt1p-positive membranes to Ypt32p-positive membranes (Rivera-Molina and Novick, 2009). Overexpression of Mon1-Ccz1 or GTPase-deficient Ypt7p inactivates the yeast Rab5 homolog, Vps21p (Rana et al., 2015). This suggests that a "counter-current" (Rivera-Molina and Novick, 2009) of Rab inactivation does operate on late endosomes. However, no interaction between a Rab5/Vps21p GAP and Rab7/Ypt7p, or a Rab7/Ypt7p effector, has yet been found.
Binding of Mon1-Ccz1 to LC3 proteins also promotes Mon1-Ccz1 function in autophagy. In yeast, Mon1-Ccz1 binds the LC3 protein Atg8p via LIR motifs in Ccz1 (Gao et al., 2018). This binding is needed for Mon1-Ccz1 association with autophagosomes. Mon1-Ccz1/Atg8p binding also activates the GEF activity of Mon1-Ccz1 activity for Ypt7p (Gao et al., 2018). Membrane recruitment may activate Mon1-Ccz1 by increasing its local concentration near membrane-associated Ypt7p, but allosteric activation upon Atg8p binding cannot be ruled out. Mon1-Ccz1 binding to metazoan LC3 proteins has not been observed, but the LIR motifs in Ccz1 are also found in mammals (Gao et al., 2018).

General Biochemical Mechanism of Rab GAP Action
Rab GAPs use the conserved Tre2/Bub2/Cdc16 (TBC) domain to activate GTP hydrolysis (Fukuda, 2011). Figure 5 shows the mechanism by which a TBC domain (green) modulates the active site of a Rab GTPase (purple). This figure depicts a homology model (Waterhouse et al., 2018) of Rab7 bound to the TBC domain of the Rab7 GAP Armus/TBC1D2A, and is based on the crystal structure of Rab33 bound to the Gyp1p TBC domain (Pan et al., 2006). A conserved glutamine from the GAP (Q713) activates a water molecule (blue) for hydrolysis. Meanwhile, a conserved arginine from the GAP (R676) provides positive charge to stabilize the GDP leaving group and the planar transition-state form of the γ phosphate, which is mimicked by an AlF 3 − molecule (Pan et al., 2006). This "dual-finger mechanism" (Pan et al., 2006) differs from the mechanism of Ras GAPs, which use a single "arginine finger" to stabilize the transition state (Scheffzek et al., 1998). This difference is important because in Ras GAP-mediated GTP hydrolysis, the hydrolyzing water is activated by a glutamine from the Ras GTPase itself (Scheffzek et al., 1998). In Rab GTPases, the equivalent glutamine is not needed for GAP-mediated GTP hydrolysis, and is flipped away from the active site in the Rab-GAP complex (Figure 5, Q67, bottom left). As a result, Rab GAPs can stimulate GTP hydrolysis by mutant Rab GTPases lacking this glutamine, though the stimulated rate is lower than that of GAP-stimulated wild-type Rab proteins by up to an order of magnitude (De Antoni et al., 2002;Pan et al., 2006).
The conserved glutamine and arginine mentioned above account for most of the GTPase-activating function of Rab GAPs. However, there are at least 27 proteins containing TBC domains in the human genome, and roughly 70 Rab GTPases. There is no satisfactory method for predicting specificity from a TBC domain's sequence. Rather, researchers have determined TBC domains' specificity empirically, by testing them for GAP activity against panels of Rab GTPases (Itoh et al., 2006;Pan et al., 2006;Fuchs et al., 2007).
Accessory domains in Rab GAPs (Figure 6) may determine Rab specificity by constraining Rab GAPs' cellular localization (Figure 2). No known Rab GAP consists of only a TBC domain. The domain architecture of Rab GAPs is diverse: they can contain lipid binding domains, coiled-coil motifs, LC3-interacting regions (LIRs), domains that bind to other small GTPases, and other domains with GAP and GEF activity (Fukuda, 2011). Some accessory domains may determine Rab GAP localization via interactions with phospholipids and other proteins (Table 1). Thus, proximity of Rab GAPs to particular Rab GTPases, which themselves have distinct cellular localizations (Li and Marlin, 2015), may dictate which Rab GAPs act on which Rabs.

Armus/TBC1D2A
The first Rab7-specific GAP to be described, Armus/TBC1D2A, was discovered via a two-hybrid screen for effectors of the Rac1 GTPase (Frasa et al., 2010). In this study, Armus was found to regulate cell-cell adhesion and E-cadherin degradation (Frasa et al., 2010). Overexpression of a construct containing the Armus TBC domain and carboxy-terminal coiled-coil motif (Figure 6)which localizes to the plasma membrane at cell-cell junctions (Figure 2) -blocks endocytosis and degradation of the cell adhesion molecule E-cadherin (Frasa et al., 2010). This construct also stabilizes cell-cell contacts in keratinocytes (Frasa et al., 2010). This stabilization may be caused by reduction in active Rab7 and a concomitant reduction in transport to the lysosome via the endocytic pathway.
Counterintuitively, siRNA-mediated knockdown of Armus expression also stabilizes cell-cell junctions (Frasa et al., 2010). Likely this is because GDI delivers Rab7 to maturing endosomes, as described above (Figure 4; Poteryaev et al., 2010). GDI binds and extracts only GDP-bound Rab proteins from membranes (Araki et al., 1990;Garrett et al., 1994). Thus, if Rab7 GTP hydrolysis is reduced via Armus knockdown, formation of the soluble Rab7-GDI complex and Rab7 delivery to endosomes also will be reduced. As evidence for the importance of GTP hydrolysis for proper Rab7 localization, knockout of another Rab7 GAP, TBC1D5, redistributes Rab7 from late endosomes to lysosomes (Jimenez-Orgaz et al., 2018). Additionally, a GTPasedeficient mutant of the yeast exocytic Rab Sec4p (Q79L) is a cold-sensitive loss-of-function allele that causes accumulation of secretory vesicles and reduced secretion (Walworth et al., 1992).
Finally, Armus and TBC-2 simply may have different functions in their respective organisms.
The Roco kinase leucine-rich repeat kinase 1 (LRRK1) also regulates Armus activity (Toyofuku et al., 2015). Autophagosome-lysosome fusion is impaired in LRRK −/− knockout cells (Toyofuku et al., 2015). Rab7-GTP levels also are elevated, and are further elevated during autophagy (Toyofuku et al., 2015). LRRK1 phosphorylation blocks an intramolecular interaction between the PH and TBC domains of Armus, though neither the binding interface nor the site of phosphorylation has yet been identified (Toyofuku et al., 2015). Thus, phosphorylation of Armus by LRRK1 may relieve an autoinhibitory interaction between its PH and TBC domains, permitting PI(3)P binding, GAP activity, or both. Interaction with another protein, perhaps via the coiled-coil domain of Armus (Figure 6), that modulates Armus membrane recruitment and/or activity also cannot be ruled out. LRRK1 is closely related to LRRK2, which is mutated in familial Parkinson's disease (Zimprich et al., 2004). LRRK2 has not been shown to phosphorylate Armus. Nevertheless, cycling of Rab7 between membrane and cytosol -which requires GTP hydrolysis by Rab7 (Araki et al., 1990;Garrett et al., 1994) is decreased in cells with Parkinson's disease-linked LRRK2 mutations (Gomez-Suaga et al., 2014).
Armus has other links to human disease. An Armus variant termed PARIS-1 was found in a screen for antigens recognized by sera from prostate cancer patients (Zhou et al., 2002). PARIS-1 is identical to Armus, except for a deletion of 11 amino acids in its TBC domain (residues 819-829). The biochemical effect of this deletion is not obvious: PARIS-1 is stably expressed (Zhou et al., 2002), and in the crystal structure of the Rab33-Gyp1p TBC domain complex used to make Figure 5, these residues do not directly contact the Rab (Pan et al., 2006). Perhaps these residues stabilize structural elements that are important for GAP activity. In another link between Armus and cancer, Armus expression is inhibited by a microRNA (miRNA) from a cluster of six miRNAs, miR-17-92/oncomir-1 (Serva et al., 2012), that is overexpressed or deleted in many cancers (He et al., 2005;Zhang et al., 2006). Finally, a genome-wide association study of multiple sclerosis (MS) patients found an association with a single nucleotide polymorphism in an intron from the gene encoding Armus (Baranzini et al., 2009). This finding has been replicated in a follow-up study of a different group of MS patients (Schmied et al., 2012). However, the basis for this linkage remains unknown.

TBC1D2B
This protein is not a splice variant of Armus, i.e., TBC1D2 or TBC1D2A (Table 1), but rather is encoded by a different gene, with 63% protein sequence identity and a virtually identical domain structure (Figure 6; Letunic et al., 2015;Sievers and Higgins, 2018). However, the Rab specificity of TBC1D2B is unknown. It has been included in this review because of its similarity to Armus.
Retromer is a five-subunit complex consisting of two subcomplexes: a cargo-binding trimer of Vps26, Vps29, and Vps35, and a membrane-binding dimer of sorting nexins (Liu, 2016). The exact set of sorting nexins that bind Vps26/29/35 is variable (Liu, 2016). Differences in sorting nexin composition affect cargo selection and the trafficking pathway in which any particular retromer complex acts (Liu, 2016). Retromer is a Rab7 and Ypt7p effector and is recruited to membranes by binding to Rab7/Ypt7p (Rojas et al., 2008;Seaman et al., 2009) and through direct membrane binding by phox homology (PX) and bin/amphiphysin/Rvs (BAR) domains in the sorting nexins (Xu et al., 2001;Carlton et al., 2004).
It should be mentioned that the TBC1D5's effect on retromer activity is not yet settled: a recent report suggests that TBC1D5 knockdown in fact boosts retromer activity (Seaman et al., 2018). This discrepancy may be due to the degree of TBC1D5 knockdown. Complete TBC1D5 knockout forces Rab7 onto lysosomes, where it cannot bind and activate the retromer (Jimenez-Orgaz et al., 2018). In contrast, incomplete TBC1D5 knockdown may allow enough Rab7 on late endosomes/MVB's to support retromer activity -and the reduction in TBC1D5 expression may in fact boost the amount of active Rab7.
How do these dual binding specificities, to LC3 proteins and the retromer, relate to one another? Experiments using purified proteins show that LC3A competes with the retromer subunit Vps29 for TBC1D5 binding (Popovic et al., 2012). In agreement with this result, TBC1D5 co-localizes with Vps35 and LC3B, but Vps35 and LC3B do not themselves co-localize (Popovic et al., 2012). Accordingly, the amino-terminal LIR motif in TBC1D5 is needed for retromer-TBC1D5 binding (Popovic et al., 2012). Autophagy triggers the switch between these alternate binding modes: upon starvation, TBC1D5-LC3A co-localization is sharply increased, whereas TBC1D5-retromer binding becomes nearly undetectable (Roy et al., 2017). This effect is autophagy-dependent and not merely triggered by starvation, which does not block TBC1D5-retromer binding in cells lacking the autophagy regulator Atg7 (Roy et al., 2017). Retromer-mediated recycling of the GLUT1 glucose transporter from endosomes to the plasma membrane is increased during starvation (Roy et al., 2017). This effect might be caused by TBC1D5-LC3A binding during autophagy (Roy et al., 2017), which sequesters TBC1D5 and permits Rab7 activation in the vicinity of the retromer.
These results have led to a model in which TBC1D15's interactions with Fis1 and LC3 proteins hold it in proximity to both mitochondria and autophagosomes (Yamano et al., 2014). In this model, TBC1D15 supports formation of autophagosomes around damaged mitochondria (Yamano et al., 2014). In the absence of TBC1D15, an excess of Rab7-GTP in apposition to mitochondria causes autophagosomes to grow larger than usual. The mechanism by which Rab7-GTP promotes autophagosome expansion is unknown. However, Rab7 knockdown in TBC1D15 −/− cells suppresses autophagosome expansion around mitochondria, suggesting that Rab7 indeed plays a role in this process (Yamano et al., 2014).
TBC1D15 also regulates mitochondrion-lysosome contacts that are not involved in mitophagy (Wong et al., 2018). These contacts are dynamic, and TBC1D15-stimulated Rab7 GTP hydrolysis is required for their termination (Wong et al., 2018). Mitochondrial fission occurs at these lysosomal contact sites (Wong et al., 2018). Expression of GTPasedeficient Rab7 mutants or GAP-deficient TBC1D15 mutants sharply reduces mitochondrial fission at lysosomal contacts and causes mitochondrial elongation (Wong et al., 2018). Thus, TBC1D15-stimulated Rab7 GTP hydrolysis regulates mitochondrial morphology even when mitophagy is not induced (Wong et al., 2018). Expression of a Fis1 mutant that cannot bind TBC1D15 also blocks mitochondrial fission at lysosomal contacts, suggesting that Fis1 mediates TBC1D15 recruitment even in the absence of mitophagy (Wong et al., 2018).
Additional interactions link TBC1D15 to other aspects of membrane trafficking. Proteomic approaches identified an interaction between TBC1D15 and Vac14, a scaffolding protein that stimulates PIKfyve, the kinase that generates PI(3,5)P 2 from PI(3)P (Schulze et al., 2014). PI(3,5)P 2 is essential for proper lysosomal pH and ion homeostasis (Hasegawa et al., 2017). The significance of the TBC1D15-Vac14 interaction is unknown, but may be a mechanism by which PI(3,5)P 2 generation is coordinated with Rab7 inactivation on lysosomes. TBC1D15 also interacts with the early endosomal Rab GTPase Rab5 (Itoh et al., 2006). However, TBCD15 does not appear to have GAP activity toward Rab5 (Itoh et al., 2006). Perhaps Rab5-TBC1D15 binding recruits a small amount of TBC1D15 to early endosomes, preventing Rab7 activation there. Finally, TBC1D15 interacts with the Rab GAP TBC1D17 (Yamano et al., 2014), though neither the target of TBC1D17 nor the role of the TBC1D15-TBC1D17 interaction has been determined.

Gyp7p
Gyp7p is the GAP for Ypt7p, the yeast vacuolar Rab GTPase (Vollmer et al., 1999;Brett et al., 2008). Deletion of the gene encoding Gyp7p has little effect on cell growth or vacuole morphology, other than a small increase in vacuole size (Brett et al., 2008). This is probably due to increased homotypic vacuole-vacuole fusion, a process that requires Ypt7p (Haas et al., 1995). Gyp7p overexpression does, however, cause vacuole fragmentation (Brett et al., 2008). Gyp7p overexpression also blocks vacuole fusion upon hypo-osmotic shock (Brett et al., 2008). Deletion of the gene for Gyp7p does not affect autophagy, though autophagy is known to require Ypt7p (Wurmser et al., 2000).
Little is known about the domain structure of Gyp7p, other than the presence of a TBC domain (Figure 6). Overall, the positioning of Gyp7p's TBC domain is similar to that of TBC1D15, toward the carboxy terminus of the protein (Figure 6). No conserved domains have been identified in the aminoterminal regions of either TBC1D15 or Gyp7p, except for TBC1D15's LIR motif (Yamano et al., 2014). This motif may be conserved in Gyp7p: the sequence of TBC1D15's LIR, from residues 280-283, is FEVI, whereas Gyp7p contains a sequence of FNDL in roughly the same region, from residues 329-332. The core LIR consensus sequence is [WFY]xx[ILV], though neighboring residues can affect LC3 family protein binding (Jacomin et al., 2016). However, Gyp7p has no known role in autophagy and is not known to bind Atg8p, the yeast LC3 protein, nor is there any known effect of mutating these residues.

Summary
The Rab7 GAPs have diverse cellular roles ( Table 1). Armus/TBC1D2A is needed for endocytosis of cell adhesion molecules and modulation of cell-cell contacts (Frasa et al., 2010). Armus also interacts with LC3 family proteins and mediates fusion of starvation-induced autophagosomes with lysosomes (Popovic et al., 2012;Carroll et al., 2013). TBC1D5 interacts with the retromer complex, a Rab7 effector, and regulates retromer's associations with membranes (Seaman et al., 2009). TBC1D5 also interacts with LC3 proteins, though it regulates autophagosome nucleation and expansion, i.e., earlier stages of autophagy than those regulated by Armus (Popovic et al., 2012;Roy et al., 2017). TBC1D15 also regulates autophagosome expansion, but in the context of mitophagy, rather than starvation-induced autophagy (Yamano et al., 2014). Like Armus and TBC1D5, TBC1D15 binds LC3 family proteins (Behrends et al., 2010;Yamano et al., 2014). TBC1D15 also binds the outer mitochondrial membrane protein Fis1, which may explain its ability to promote assembly of autophagosomes around damaged mitochondria (Onoue et al., 2013). In addition, TBC1D15 regulates dynamic lysosomemitochondrion contacts that mark the sites of mitochondrial fission (Wong et al., 2018). Finally, Gyp7p is the GAP for the yeast Rab7 homolog Ypt7p (Vollmer et al., 1999;Brett et al., 2008). Little is known about Gyp7p, perhaps because phenotypes for its deletion or overexpression are fairly weak (Brett et al., 2008). No interactors for Gyp7p, other than Ypt7p, have been identified, nor does Gyp7p have any known role in autophagy.

BACTERIAL EFFECTORS/VIRULENCE FACTORS THAT MODULATE RAB7 NUCLEOTIDE BINDING
Bacterial pathogens manipulate host intracellular trafficking in order to establish comfortable intracellular niches (Weber and Faris, 2018). This is accomplished by secreted bacterial proteins termed virulence factors or effectors (Alix et al., 2011). These effectors allow bacterial cells, engulfed via phagocytosis, to replicate inside host cells. Rab GTPases, including Rab7, are common targets of bacterial effector proteins (Stein et al., 2012). Two bacterial effectors manipulate the nucleotide-binding state of Rab7: RidL from Legionella pneumophila, and SopD2 from Salmonella enterica (serovar Typhimurium).
RidL is secreted by L. pneumophila, which causes Legionnaires' Disease (Finsel et al., 2013). RidL is a substrate for the Legionella type IV secretion system (T4SS), which secretes bacterial proteins into host cell cytosol, across the bacterial inner and outer membranes and the limiting membrane of the Legionella-containing vacuole (LCV) that contains intracellular Legionella (Backert and Meyer, 2006). After secretion, RidL localizes to endosomes, where it binds the retromer via Vps29 (Figure 7A; Finsel et al., 2013). This interaction competitively blocks TBC1D5-Vps29 binding and displaces TBC1D5 into the cytosol (Barlocher et al., 2017;Yao et al., 2018). RidL does not displace Rab7 from LCVs; thus, Rab7 remains GTPbound in the presence of RidL (Figure 7A; Barlocher et al., 2017). How RidL promotes Legionella survival and replication remains unclear. Perhaps the LCV acts as a sink for Rab7-GTP, reducing Rab7 levels on late endosomes and blocking transport of lysosomal hydrolases from late endosomes to LCVs.

Summary
Two known bacterial effectors modulate the nucleotide-binding state of Rab7. Legionella RidL displaces TBC1D5 from the retromer and thus probably locks Rab7 in its GTP-bound form. Salmonella SopD binds directly to GDP-bound Rab7 and blocks nucleotide exchange, i.e., activation. It is likely that more examples of the manipulation of Rab7 nucleotide binding by bacterial effectors will be found. Individual Legionella strains each secrete an estimated 300 proteins via the T4SS (Schroeder, 2017). Between the many Legionella strains that have been isolated, roughly 600 distinct T4SS substrates have been identified, and of these only 7 are conserved among all strains (Burstein et al., 2016). The repertoire of the Salmonella T3SS is more limited, with just 30 known substrates, but only about half of these have known functions (Figueira and Holden, 2012).

CONCLUSION AND OUTLOOK
Rab7 and Ypt7p govern both endolysosomal trafficking and autophagy (Guerra and Bucci, 2016). They accomplish this by binding to a large and diverse set of effectors (Seals et al., 2000;Wurmser et al., 2000;Cantalupo et al., 2001;Jordens et al., 2001;Lazar et al., 2002;Pankiv et al., 2010;Sun et al., 2010). But before this can happen, the nucleotide-binding state of Rab7 and Ypt7p must be controlled in both time and space by the GEF and GAPs discussed here. Much work has gone into discovering these factors and elucidating their protein-protein and protein-lipid interactions (Figures 4, 6 and Table 1). These studies have made it possible to now seek a deeper level of understanding: How do Rab7/Ypt7p's GEF and GAPs specify Rab7/Ypt7p activation at particular places and times?
Answers to these questions may come from an improved understanding of how the intermolecular interactions of Rab GEFs and GAPs change in time and space, and how they are affected by cellular metabolic state and the physical and chemical growth environment. Methods that could prove useful include FRET-based and split-enzyme/split-fluorescent protein biosensors for protein-protein interactions (Kerppola, 2006), proximity ligation and proteomics for unbiased sampling of protein-protein binding (Lobingier et al., 2017), and ion mobility mass spectrometry for isolating and characterizing protein-lipid complexes from native environments (Liu et al., 2017). Such approaches will allow development of quantitative, integrated models that explain how Rab7 nucleotide binding is regulated and, in turn, regulates endolysosomal trafficking and autophagy.

AUTHOR CONTRIBUTIONS
CS wrote the manuscript and prepared the figures.