The autophagic roles of Rab small GTPases and their upstream regulators: a review.

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The autophagic roles of Rab small GTPases and their upstream regulators a

Zsuzsanna Szatmári & Miklós Sass

a

a

Department of Anatomy, Cell and Developmental Biology; Eötvös Loránd University; Budapest, Hungary Published online: 04 Jun 2014.

Click for updates To cite this article: Zsuzsanna Szatmári & Miklós Sass (2014) The autophagic roles of Rab small GTPases and their upstream regulators, Autophagy, 10:7, 1154-1166, DOI: 10.4161/auto.29395 To link to this article: http://dx.doi.org/10.4161/auto.29395

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ReviewReview Autophagy 10:7, 1154–1166; July 2014; © 2014 Landes Bioscience

The autophagic roles of Rab small GTPases and their upstream regulators A review

Zsuzsanna Szatmári* and Miklós Sass

Downloaded by [Michigan State University] at 17:40 09 February 2015

Keywords: autophagy, GAP, GEF, RAB, small GTPases Abbreviations: ATG, autophagy-related; COG, conserved oligomeric Golgi; EE, early endosome; ER, endoplasmic reticulum; GAP, GTPase activating protein; GAS, Group A Streptococcus; GDP, guanosine diphosphate; GEF, guanosine nucleotide exchange factor; GTP, guanosine triphosphate; HD, Huntington disease; HOPS, homotypic fusion and vacuolar protein sorting complex; (M)TOR, (mechanistic) target of rapamycin; (M)TORC1, (mechanistic) TOR complex 1; PAS, phagophore assembly site; PD, Parkinson disease; PtdIns3P, phosphatidylinositol 3-phosphate; PIK3C3, class III phosphatidylinositol 3 kinase; RE, recycling endosome; SNARE, SNAP (soluble NSF attachment protein) receptor; TRAPP, transport protein particle; ULK, unc-51 like autophagy activating kinase; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting

Macroautophagy is an evolutionarily conserved degradative process of eukaryotic cells. Double-membrane vesicles called autophagosomes sequester portions of cytoplasm and undergo fusion with the endolysosomal pathway in order to degrade their content. There is growing evidence that members of the small GTPase RAB protein family—the well-known regulators of membrane trafficking and fusion events—play key roles in the regulation of the autophagic process. Despite numerous studies focusing on the functions of RAB proteins in autophagy, the importance of their upstream regulators in this process emerged only in the past few years. In this review, we summarize recent advances on the effects of RABs and their upstream modulators in the regulation of autophagy. Moreover, we discuss how impairment of these proteins alters the autophagic process leading to several generally known human diseases.

Introduction The genetic regulation of macroautophagy (hereafter autophagy) was originally described in the yeast Saccharomyces cerevisiae, but it has also an important role in multicellular organisms as a cytoprotective response to stress and pathological conditions.1 The process is characterized by the formation of doublemembrane autophagosomes arising from a phagophore assembly site (PAS). The PAS is a well-defined cytoplasmic site marked by a subset of autophagy-related (ATG) proteins.2 The formation of the phagophore from the PAS requires the subsequent activity of *Correspondence to: Zsuzsanna Szatmári; Email: [email protected] Submitted: 01/21/2014; Revised: 05/19/2014; Accepted: 05/28/2014; Published Online: 06/05/2014 http://dx.doi.org/10.4161/auto.29395

the ULK1/2 (Atg1 in yeast) kinase complex, which, together with the class III phosphatidylinositol 3-kinase (PIK3C3/VPS34) complex initiates the phagophore nucleation and expansion and 2 ubiquitin-like conjugation systems: the ATG12–ATG5ATG16L1 complex and the phosphatidylethanolamine-conjugated LC3/Atg8. Completed autophagosomes undergo fusion events with various members of the endolysosomal system to form amphisomes or autolysosomes and in either case their content is degraded by lysosomal hydrolases.3 RAB proteins, members of the RAS GTPase superfamily, are key regulators of membrane trafficking and fusion events.4 All of the RABs contain a conserved nucleotide binding domain which is able to bind both GTP and GDP. RABs generally cycle between GTP-bound active and GDP-bound inactive forms (Fig. 1). In their active state, RAB proteins recruit various effectors to the membrane, which they are attached to. RABs have a low intrinsic hydrolase activity; their hydrolysis rate depends on GTPase-activating proteins (GAPs). These proteins can complement the catalytic site of RABs, thereby promoting GTP hydrolysis. Inactive, GDP-bound RABs are removed from their target membrane and kept soluble in the cytoplasm by GDP dissociation inhibitors (GDIs). Upon RAB activation, GDIs are removed mainly by GDI displacement factors (GDFs). Thereafter, guanine nucleotide exchange factors (GEFs) activate RAB proteins by stimulating the exchange of GDP to GTP. Active RABs can stably attach to membrane surfaces via their C-terminal lipid (geranylgeranyl) anchor, provided by RAB escort proteins. In mammals, there are more than 70 known RAB proteins among which about 10 have a defined function in autophagy (Table 1). However, only a few of their upstream regulators were implicated in this process. Here we summarize recent advances on the role of RABs and their regulators in autophagy, showing their primary functions and highlighting many well-known GEFs and GAPs whose autophagic function needs further investigation. Furthermore, we discuss how impaired RAB cascades

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Department of Anatomy, Cell and Developmental Biology; Eötvös Loránd University; Budapest, Hungary


can cause alterations in the autophagic process leading to various human diseases.

Endosomal RABs: RAB4, RAB5, and RAB7 In parallel with autophagy, the endosomal system also provides macromolecules and energy sources for the cell. Cargo taken up by different ways of endocytosis is subsequently transported to early or sorting endosomes (EEs). RAB5 is a well-known marker of these structures: active RAB5 recruits effector proteins, which play a role in maintaining, trafficking, cargo recycling, and maturation of EEs. RAB5 is involved in vesicle fusion events as well: its yeast ortholog, Vps21, mediates homo- and heterotypic fusion of EEs with endocytic carriers through its effectors CORVET and EEA1/Vac1.53 Furthermore, RAB5 participates in the endosomal recruitment of PIK3C3/VPS34, which in turn produces phosphatidylinositol 3-phosphate (PtdIns3P) on the EE membrane.54 Lately, RAB5 was found to be a regulator of the early steps of autophagosome formation. Similarly to its endosomal functions, RAB5 may have a role in the recruitment of the autophagic BECN1-PIK3C3 complex and subsequent PtdIns3P production on the phagophore membrane. A previous work of Ravikumar and colleagues showed that RAB5 regulates the conjugation of ATG12 to ATG5 through its effector, PIK3C3, as a part of the autophagic BECN1 complex.13 This study suggested a direct role for RAB5 in the process of autophagosome formation, since its effect on autophagy was not due to its endocytic roles or through the regulation of MTOR signaling. Moreover, both RAB5 and PIK3C3 were found to be required for the formation of autophagosomes during autophagy induced by the nonstructural protein 4B (NS4B) of hepatitis C virus.14 Interestingly, recent studies showed that PIK3CB/p110-β, the catalytic subunit of the class IA phosphoinositide 3-kinase complex facilitates this RAB5 function.15,16 In these studies, Dou and colleagues showed that loss of PIK3CB leads to impaired autophagy. They found that following growth factor limitation, PIK3CB dissociates from the growth factor receptor complex and associates with RAB5, positively regulating its activity and enhances its interaction with PIK3C3, thereby promoting autophagosome formation.

RAB4 localizes to early endosomes and mediates cargo recycling toward the plasma membrane on the so-called fast recycling route.55,56 In addition, RAB4 was recently found to have a role in the early steps of autophagy.18 This study showed an increased colocalization of RAB4 with LC3 due to autophagy induction by starvation or rapamycin treatment. Talaber and colleagues also showed that a C-terminally truncated native isoform of RAB4 shows an enhanced colocalization with LC3 and mitochondria and its expression leads to an increased formation of autophagic structures. Furthermore, overexpression of functional RAB4 protein results in the accumulation of mitochondria during autophagy induction, suggesting that it promotes the formation not only of autophagosomes, but also of the mitochondrial network, thereby preserving mitochondria upon autophagy induction. Whether RAB4 plays a direct role in the early stages of autophagy or acts indirectly is not understood yet, since the exact molecular mechanism through which RAB4 can orchestrate autophagy is still poorly characterized. After cargo recycling, EEs mature into late endosomes (LEs).57 During the maturation process, inactivated RAB5 is substituted by RAB7 on the endosomal membrane. Subsequently, RAB7 recruits its effectors which are required for LE trafficking and fusion with lysosomes.58 Two well-known RAB7 effectors, RILP (Rab-interacting lysosomal protein) and OSBPL1A/ ORP1L (oxysterol-binding protein-like 1A) are necessary for the minus end-directed transport of late endosomes along microtubules.59-61 These proteins bind to active RAB7 simultaneously;

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Figure 1. GTP-GDP exchange cycle of RAB proteins. RABs cycle between GTP-bound active and GDP-bound inactive forms. In their active, membrane-attached state they recruit various effectors. GTPase-activating proteins (GAPs) increase the GTP hydrolysis rate, thereby inactivating RABs. Inactive RABs are sequestered in the cytosol by GDP dissociation inhibitors (GDIs). Upon RAB activation, GDI displacement factors (GDFs) can displace GDIs. Afterwards, guanine nucleotide exchange factors (GEFs) activate RAB proteins by changing GDP to GTP.

Categories based on the primary roles of RABs

RABs

Primary localization/role

Role in autophagy

References

Golgi gatekeepers

RAB1

ER-Golgi transport

Phagophore assembly by regulating ATG9 localization;

5, 6–10, 11

RAB5

Early endosomes

RAB4

Early endosomes, cargo recycling

Endosomal RABs


RAB7

Further RABs implicated in autophagy

Other Golgi RABs

Endosomal RABs/ Golgi gatekeepers

RAB8

RAB9

Late endosomes

Transport of secretory and recycling vesicles toward plasma membrane

Recycling from late endosomes to TGN

Regulation of TORC1 activity

12

Regulation of PIK3C3-BECN1 complex

13–16


12, 17

Autophagosome formation

18

Microtubular transport of autophagosomes

19–21

Autophagosome maturation

22–24, 25–31

Autophagic lysosome reformation

32

Autophagosome formation

33–35


36

Autophagy-based secretion

37

Autophagosome maturation during antimicrobial autophagy

38

Autophagosome maturation during antimicrobial autophagy

39

Autophagosome formation during ATG5- and ATG7-independent noncanonical autophagy

40

Providing RE-derived membrane source for autophagy

41–43

Maturation of autophagosomes

44–47

RAB11

Secretory pathway (yeast), recycling endosomes


12

Further RABs implicated in autophagy

RAB24

ER, cis-Golgi

Colocalization with LC3 upon autophagy-induction

48

RAB32

Biogenesis of lysosome-related organelles, regulation of ER-mitochondria contact

Required for autophagosome formation

49, 50

Other Golgi RABs

RAB33

Medial Golgi

Role in autophagosome formation and maturation

51, 52

RAB11 has dual role: it can be categorized either as an endosomal or a Golgi gatekeeper RAB protein.

their interaction facilitates the association of SPTBN2/βIII spectrin and the dynein-dynactin complex with the endosomal membrane, which in turn mediates the microtubule-attached vesicle movement. Furthermore, RAB7 through the homotypic fusion and vacuole protein sorting (HOPS) complex and its associated SNAREs62,63 and through RNF115/Rabring7 (ring finger protein 115/RAB7-interacting RING finger protein),64 plays a key role in the late endosome-lysosome fusion. In addition, the BECN1-binding UVRAG (UV radiation resistance associated), while binding class C VPS members of the HOPS complex, also enhances RAB7 activity.65 Accordingly, KIAA0226/Rubicon acts as a negative regulator of endosome maturation by sequestering UVRAG from the class C VPS complex and preventing it from RAB7 activation.22,23,66,67 During autophagosome maturation RAB7 acts similarly as in endosome maturation (Fig. 2).24 However, while OSBPL1A has not yet been implicated in autophagy, RAB7-RILP interaction was recently found to be required for neuronal autophagy.19 This study showed that during neuronal stress, the presence of IGF1 enhances the interaction of RAB7 with RILP, thereby facilitating autophagic flux. A further study showed that RAB7 together with its LC3-interacting effector FYCO1 (FYVE and coiled-coil

domain containing 1) participates in the microtubular transport of autophagosomes.20 FYCO1 is observed on endosomes and autophagosomes as well, where it forms a complex and colocalizes with RAB7. Loss of FYCO1 results in the accumulation of autophagosomes in the perinuclear region of the cells, suggesting that it plays a key role in plus end-directed microtubule transport of autophagosomes. These findings suggest that RAB7 and its effectors can directly mediate autophagosome trafficking on microtubule tracks in both directions. Supporting this, a recent study showed that autophagosomes at the early stage of their maturation process can move in neurons using either kinesin or dynein motors. After a while, however, they show unidirectional movement toward the cell soma using only dynein motor proteins.21 In HeLa cells only plus-end directed transport of autophagosomes is observed, and all evidence of minus-end directed autophagosomal transport and bidirectional movement are derived from neuron models; it is possible that these latter types of autophagosome trafficking is a specific features of neurons. Similarly to its endosomal functions, RAB7 is required for fusion of autophagosomes with late endosomes or lysosomes.25-28 These studies show that RAB7 colocalizes with LC3 and its silencing or overexpression of the GDP-locked, dominant negative

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Table 1. RAB proteins implicated in autophagy

Figure 2. Various roles of RAB7 in autophagy. Through its interaction partners, RAB7 is implicated in the bidirectional transport of autophagosomes. RILP participates in the recruitment of dynein-dynactin complex, whereas FYCO1 interacts with kinesins. Other RAB7 effectors, UVRAG, KIAA0226/Rubicon, and RNF115 have a role in autophagosome maturation. While the HOPS complex associated SNAREs, VTI1B, and VAMP8, are involved in autophagosome-lysosome fusion, the HOPS itself, although a well-known RAB7 effector, is not implicated in this process. Furthermore, RAB7 is implicated in the regulation of TORC1 activity, and, together with its effector PLEKHF1/Phafin1, is also described in autophagosome (AP) formation.

mechanism is poorly understood yet. Similarly, 2 recent studies showed that RAB7 is required at the initial steps during the antibacterial autophagic response against GAS, since depletion of RAB7 leads to a decrease in formation of GAS-containing autophagic vacuoles.34,35 Further studies indicated that RAB5 and RAB7 play a role even in the activation of the main nutrient and energy sensor of eukaryotic cells, MTORC1, which controls the balance of anabolic and catabolic processes and is one of the most important regulators of autophagy. The core member of this complex, MTOR, blocks autophagy induction through the inhibitory phosphorylation of several autophagic proteins.75 Recently, MTORC1 was shown to be activated at the surface of lysosomes by an amino acid-sensing protein cascade (reviewed in ref. 76). In agreement with this, another study found that intact late endosomes are essential for MTORC1 signaling, revealing a novel function of RAB7 as a required regulator of MTOR activity.36 Two studies proposed a similar role for RAB5 in regulation of TORC1 activity and localization. Li and colleagues showed that either knockdown or uncontrolled expression of wild-type, constitutively




RAB7 lead to an increase in size and number of autophagosomes due to their impaired fusion with lysosomes. Supporting these findings, a recent study revealed a similar role for RAB7 in selective autophagy as well.29 Although some results show that the HOPS complex-associated SNAREs VTI1B and VAMP8 play a role in this fusion process,30 the HOPS member VPS16 was not found to be necessary for it.31 Furuta and colleagues found that during antimicrobial autophagy induced by Streptococcus pyrogenes (also known as Group A Streptococcus, GAS) infection, VTI1B, and VAMP8 colocalize with LC3 on autophagic structures and these SNAREs are required for elimination of bacteria through mediating the fusion of GAS-containing xenophagosomes with lysosomes.30 Furthermore, several studies indicated that UVRAG and KIAA0226/Rubicon also have an effect on autophagic maturation. These studies indicate that UVRAG physically interacts with the HOPS complex, thereby facilitating RAB7 activity and the maturation process of both endosomes and autophagosomes.65,66 In contrast, KIAA0226 inhibits PIK3C3 activity and downregulates autophagy, probably through its interaction with RAB7.22,67 However, our growing knowledge on the convergence of endosomal and autophagic routes raises the possibility that these RAB7 effector proteins have only an indirect impact on the late stages of autophagy.68 There are several lines of evidence that closed autophagosomes fuse not only with lysosomes, but also with various populations of endosomes69-71 to form hybrid organelles called amphisomes.72 In many cell types amphisome formation seems to be essential for subsequent lysosomal fusion and degradation. Thus, in these cells the presence of mature endosomes is required for the completion of the autophagic process. Consequently, it is possible that impairment of autophagic maturation due to depletion of RAB7 or UVRAG is caused in fact by the lack of mature endosomes. Supporting this, recent studies showed that direct autophagosome-lysosome fusion is mediated independently of RAB7 and its effectors, by the SNARE protein STX17 (syntaxin 17).73,74 All of these studies suggest that RAB7 effectors facilitating endosome maturation have a cell typedependent role in autophagy. There are only a few studies revealing a role for RAB7 in other stages of autophagy. A former paper indicated that MTOR (mechanistic target of rapamycin) regulates the process of autophagic lysosome reformation likely through RAB7.32 Yu and colleagues found that upon prolonged autophagy, reactivation of MTOR is responsible for the generation of protolysosomal tubules, which subsequently mature into functional lysosomes. They showed that GTP hydrolysis by RAB7 is required for this process, since treatment of cells with a nonhydrolizable GTP analog results in the presence of enlarged, long-lasting autolysosomes. In addition, through its effector PLEKHF1/Phafin1,33 RAB7 is also implicated at an early step, at the formation of autophagosomes. This study reported that PLEKHF1 is recruited to lysosomes by RAB7, based on its colocalization with the lysosomal marker LysoTracker Red. It was also found that overexpression of PLEKHF1 results in the accumulation of LC3-positive autophagosome-like structures, suggesting that PLEKHF1 has a role in the induction of autophagosome formation, but the underlying

RABs

RAB1/ Ypt1

Role in autophagy

GAP

Role in autophagy

-

Gyp1

-

TRAPPII

-

TRAPPIII

6-9

Role in autophagosome formation

Gyp5

-

Gyp8

-

TBC1D20

-

VPS9D1/Vps9

-

Msb3

-

RABGEF1/RABEX5

Its knockdown leads to enhanced autophagy77

RN-TRE

-

RIN1

-

RIN2

-

RIN3

-

ALS2/Alsin

Role in autophagosome maturation78

ALS2CL

-

GAPVD1/RME-6

-

RAB7/ Ypt7

MON1-CCZ1

-

Msb3

-

Gyp7/TBC1D15

-

RAB8

RAB3IP/Rabin8

-

TBC1D17

-

RAB9

DENND2A

-

TRAPPII

-

EVI5

-

HTT complex

-

Claret

Involvement in autophagy50

RUTBC1

-

HPS/BLOC-3

TBC1D25/OATL1

Involvement in autophagosome maturation52

SGSM2/RUTBC1

-

?

TBC1D14 (putative GAP)

Role in autophagosome formation41

?

TBC1D5

LC3-binding, required for autophagy124

RAB5/ Vps21


GEF TRAPPI

RAB11 RAB32 RAB33

active or dominant negative forms of Rab5 significantly inhibits TOR activity in Drosophila S2 cells, potentially through the regulation of the amino acid sensing RAG GTPase complex.12 Meanwhile Bridges and others found that disruption of RAB5 impairs TOR activity and localization in yeast and mammalian cells.17 Their study suggests that RAB5 carries out this role through acting on PIK3C3 kinase and its PtdIns3P production. All of these results indicate that fine-tuning of RAB cascades is important for their proper autophagic function. Although RAB4, RAB5, and RAB7 were implicated in various autophagic roles and several of their GEFs and GAPs are known, only 2 of these proteins were studied in the context of autophagy and implicated in the regulation of this process (Table 2). A recent study showed that in the nematode Caenorhabditis elegans the siRNA-mediated silencing of a wellknown RAB5 GEF, RABGEF1/RABEX5 results in an enhanced autophagy.77 Disruption of the ubiquitin-binding domain of RABGEF1 is responsible for autophagy induction, suggesting that this motif can be responsible for switching the involvement of RABGEF1 between autophagy and the endocytic pathway. Another RAB5 GEF protein, ALS2/Alsin, was observed as a key participant of late stages of autophagy.78 Missense mutation of ALS2 leads to decreased amphisome formation and impaired

autophagic degradation. However, it is possible that ALS2 has only an indirect impact on autophagy through its role in endosome maturation. As endosomal RABs have important functions in autophagy regulation, clarifying and further investigating their GAP and GEF proteins in this process can be essential for understanding the distinct roles of RAB5 and RAB7 cascades in orchestrating autophagy.

The Golgi Gatekeepers: RAB1 and RAB11 Yeast Ypt1 and its mammalian homolog RAB1 mediate the vesicular transport between the endoplasmic reticulum (ER) and Golgi apparatus. RAB1 participates in vesicle trafficking and— through its interaction partners—facilitates tethering and subsequent fusion of ER-derived vesicles with the cis-Golgi.79 Recently, RAB1 was described as an important factor of autophagosome formation as well, since depletion of functional RAB1 protein leads to an impairment in this process.5 Several studies revealed the existence of special ER sites where the phagophore is formed from a potentially ER-derived membrane source.80-82 Upon autophagy induction, ATG proteins are recruited to these PAS sites in a well-defined order and mediate

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Table 2. Rab GEFs and GAPs and their role in autophagy

Figure 3. RAB1/Ypt1 has a key role in autophagosome formation. Through its interactors, the conserved oligomeric Golgi (COG) complex and Atg11, it facilitates ATG9/Atg9 cycling. Moreover, activated by the RAB1 GEF, TRAPPIII, RAB1 participates in the recruitment of ULK1/Atg1 to the PAS.

and COG, both of which participate in the regulation of Atg9dependent membrane transport to the PAS, thereby providing a membrane source for the forming autophagosomes. Parallel with these, Ypt1 recruits to the PAS Atg1 kinase, a core component of autophagy induction complex, which is required for the regulation of phagophore formation and expansion. Ypt31/32, the yeast homologs of RAB11 have a key role in trafficking of secretory vesicles from the Golgi to the plasma membrane,92 whereas RAB11 is a widely used marker of recycling endosomes (REs) in mammals and is required for cargo transport from REs toward the plasma membrane.93 As was mentioned above, ATG9-marked vesicles can derive not only from Golgi, but also from REs: Longatti and colleagues found that ATG9 localizes to RAB11-positive REs.41 This study revealed a role for the RAB GAP TBC1D14 as an effector of RAB11 in the early steps of autophagosome formation (Fig. 4). The study showed that TBC1D14 binds ULK1 kinase and this interaction is responsible for the recycling endosomal localization of ULK1. Furthermore, loss of RAB11 results in impairment of TBC1D14driven RE tubulation and autophagosome formation. All of these data suggest that REs are a possible membrane source of autophagy and RAB11 is required for the transport of RE-derived membrane to the autophagosome precursors. A similar role for RAB11 was suggested in a recent work in Drosophila melanogaster showing that RAB11, together with the Drosophila homolog of sorting nexin 18 (SNX18), participates in membrane traffic from REs to the autophagosome formation sites, providing a RE-derived membrane source for phagophore expansion.42 Moreover, Puri and colleagues demonstrated that several autophagic membrane sources converge at the level of REs and the fusion of different populations of plasma membrane-derived vesicles is essential for autophagosome formation.43 They showed that ATG9 participates in membrane transport from ATG16L1decorated autophagic precursor vesicles to the REs, where these carriers fuse with endosomes in a SNARE-dependent manner.




the subsequent formation and completion of autophagosomes.83 First, the members of the ULK1/2-RB1CC1 (Atg1-Atg17 in yeast) complex gather to the ER-associated autophagic sites. This is followed by the recruitment of the ATG14-containing BECN1-complex and PtdIns3P production due to PIK3C3 activity. In yeast, Atg17 functions as a scaffold protein during autophagosome biogenesis.84 Upon autophagy induction it binds to Atg29 and Atg31. This interaction is required for directing Atg9decorated vesicles to the PAS and for phagophore organization. ATG9/Atg9, the only known transmembrane protein required for autophagosome formation, primarily localizes to recycling endosomes and small Golgi-derived vesicles, shuttling between peripheral cytoplasmic sites and the PAS, and also participates in providing a membrane source for autophagosomes.85,86 Recently, Wang and colleagues found that Atg17 directly binds to the well-known RAB1/Ypt1 GEF, TRAPPIII (transport protein particle III) thereby recruiting it to the PAS.6 Once TRAPPIII is bound to Atg17, it activates Ypt1, which subsequently recruits Atg1 to the PAS. The specificity of Atg1 recruitment is indicated by the observation that overexpression of Ypt1 results in a significant increase in the presence of Atg1 at the PAS and in enhanced autophagy, while there is no detectable change in the amount of other Atg proteins on the phagophore. However, while in yeast there are 3 TRAPP complexes in existence, only TRAPPIII and its specific component, Trs85 were found to be involved in the early stages of both nonselective autophagy and pexophagy, the selective engulfment of peroxisomes.7 In line with these findings, Meiling-Wesse and colleagues showed that lack of Trs85 lead to defects both in autophagosome formation and the cytoplasm to vacuole targeting (Cvt) pathway, since recruitment of Atg8 is impaired in these mutant yeast cells.8 In addition, a further study reported that Trs85 is responsible for the PAS targeting of TRAPPIII and the subsequent recruitment and activation of Ypt1.9 In a recent study, Atg11 was identified as an effector of the yeast Ypt1.10 Atg11, a scaffold protein at the PAS, is involved in different types of selective autophagy, for instance in the Cvt pathway.87 Moreover, Atg11 interacts with Atg9 and regulates its cycling through the PAS during selective autophagy.88 In addition to these results, the conserved oligomeric Golgi (COG) complex, another known effector of Ypt1,89 is also implicated in autophagy.11 This study showed that COG localizes to the PAS where it interacts with Atg proteins. Furthermore, COG is required for the proper subcellular localization of Atg9. Supporting these results, RAB1 is required for selective autophagy in higher eukaryotic cells as well: it was described as a key regulator of Salmonella clearance in mammalian cells.90 A further study indicated a role for RAB1A in homeostasis of SNCA/α-synuclein the major component of Lewy bodies (protein aggregates specific for Parkinson disease).91 Taken together, these studies suggest a key role for RAB1/ Ypt1 in the organization of the very early steps of autophagy (Fig. 3). Once Atg17—the most upstream factor in the regulation of autophagosome formation—is attached to the PAS, besides other ATG proteins it recruits the TRAPPIII complex, which subsequently activates Ypt1. Through its effector proteins, Ypt1 orchestrates autophagosome biogenesis: it recruits Atg11

They observed the correlation of these fusion events with the rate of autophagosome formation and upon autophagy induction they found an increased fusion of vesicles serving as a membrane source of autophagosomes and a decreased recycling, suggesting that this mechanism has a role in the regulation of early autophagic steps. Taken together these results suggest that RAB11 may directly contribute to autophagosome formation through providing various membrane sources for the phagophore. In addition to these, both Rab1 and Rab11 were described as required factors for TORC1 activity in Drosophila, since knockdown of these proteins decreases significantly the phosphorylation of RPS6KB/S6K, a well-known TOR target.12 Interestingly, besides autophagosome formation, RAB11 seems to play an important role in another stage of autophagy. It was previously shown that RAB11 is required for the fusion of autophagosomes with multivesicular bodies (MVBs).44-46 These studies reported that depletion of RAB11 results in the decreased convergence of RAB11-decorated MVBs with LC3-labeled autophagosomes. Furthermore, this role of RAB11 may be affected by the toxic aggregates of the HTT/huntingtin protein during Huntington disease (HD), which can lead to neurodegeneration. In line with these studies, our latest work revealed a role for Rab11 in amphisome formation in Drosophila, since loss of functional Rab11 protein resulted in the accumulation of autophagosomes and enlarged, acidic late endosomes.47 Our results suggest that upon autophagy induction Rab11 translocates from REs to autophagosomes labeled with Atg8a, where it physically interacts with the microtubule binding protein Hook, a negative regulator of endosome maturation. This interaction presumably prevents Hook from anchoring late endosomes to microtubules thereby allowing subsequent endosome-autophagosome fusion. These data suggest that upon autophagy induction, when the maturation of autophagosomes requires an increased input from the endolysosomal system, Rab11 can facilitate endosome

maturation by regulating the subcellular localization of Hook (Fig. 4). Although, both RAB1 and RAB11 have several known upstream regulators, only the RAB1 GEF TRAPPIII was described as a player in autophagy (Table 2). TRAPP complexes are common GEFs of the yeast homologs of RAB1 and RAB11, Ypt1 and Ypt31/32, respectively.94,95 Moreover, these small GTPases were found in the same Rab cascade in yeast: the GEF of Ypt32 is a putative effector of Ypt1.96 In addition to this, Ypt32 facilitates the proper localization and activity of the Ypt1 GAP, and Gyp1.97 Taken together, these results suggest the existence of a Rab conversion mechanism in the yeast secretory pathway, during which Ypt1 is substituted with Ypt32. The close relationship of these 2 proteins and their involvement at the same stage of autophagy raises the possibility that more of their upstream regulators may have a role in this process, but this question requires further investigation.

The Other Golgi RABs: RAB9 and RAB33 RAB9 functions at the level of late endosomes in cargo recycling toward the trans-Golgi network (TGN).98 Similarly to RAB7, RAB9 plays a role in the autophagic clearance of GAS.39 RAB9A is recruited to matured GAS-containing autophagosomes and is crucial for homotypic and lysosomal fusion of these structures. Supporting these findings, depletion of RAB9A lead to the impaired degradation of GAS. A former study found that RAB9, facilitating the fusion of TGN and late endosome-derived vesicles, is required for autophagosome formation during the process of ATG5- and ATG7-independent noncanonical autophagy.40 This study showed that RAB9 localizes to LAMP2-positive autolysosomes. Knockdown of RAB9 leads to a decrease in the number of autophagic structures, but increases

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Figure 4. Roles of RAB11 in the regulation of autophagy. RAB11 seems to have different roles in distinct steps of autophagy. Together with TBC1D14 it plays a role in membrane trafficking from the recycling endosome (RE) to the PAS. SNX18 is also implicated in this process. Furthermore, upon autophagy induction by starvation, RAB11 removes Hook—a negative regulator of endosome maturation—from LEs allowing subsequent fusion of a LE with an autophagosome (AP).

Further RABs involved in Autophagy: RAB8, RAB24, and RAB32 RAB8 has a well-defined role in vesicle transport toward the plasma membrane both in constitutive biosynthetic and RAB11-independent recycling pathways.101-103 Similarly to these functions, RAB8A was recently implicated in autophagy-based unconventional secretion the of proinflammatory cytokine, IL1B.37 This study showed that autophagy-induction enhances the secretion of IL1B. This process depends on inflammasomes, ATG5, CTSD/cathepsin D, and RAB8A as well. Furthermore, through its effector, the innate immunity regulator TBK1 (TANK-binding kinase 1), RAB8B appears to have a role in the maturation of Mycobacterium-containing autophagosomes.38 TBK1 is recruited to autophagic structures by RAB8B and it facilitates selective antibacterial autophagy through activating the autophagic adaptor protein SQSTM1/p62 by phosphorylation. How RAB8 is recruited to autophagosomes and whether it has other roles in the regulation of selective or nonselective autophagy requires further investigation. RAB24 is detected on ER, cis-Golgi and their intermediate compartment and it colocalizes with the autophagy marker LC3.104 The frequency of this colocalization increased upon starvation-induced autophagy and, moreover, overexpression of RAB24 results in an increased number of autophagic structures.48 This observation suggests that RAB24 has a role in autophagy; however clarifying its exact function needs further research.

RAB32 has a key role in the biogenesis of lysosome-related organelles, such as melanosomes105 and it was recently described as a regulator of ER-mitochondria interaction at mitochondriaassociated membrane sites106 and also as a required factor for autophagosome formation.49 This study reported that overexpression of RAB32 leads to an increase in the number of LC3-positive autophagic structures. They also found the ER localization of RAB32 to be required for autophagosome formation, suggesting that it has a role in the very early steps of autophagy by providing an ER-derived membrane source for the phagophore. In line with these, depletion of RAB32 results in the impaired autophagic clearance of SQSTM1-attached ubiquitinated proteins. These findings are in line with the recent observations of Hamasaki and colleagues, which suggested that autophagosome formation can occur at ER-mitochondria contact sites.107 Taken together, these results raise the possibility that RAB32 may have a role in this mechanism. Supporting these findings, a further study showed that RAB32 and its GEF, Claret, are required for normal size of lipid droplets and also for autophagy in Drosophila.50

Autophagic RABs in Pathological Conditions Several RAB proteins described in the previous sections are implicated in various acquired and genetic diseases. Impairments in regulation or proper function of these proteins can result in autophagic failures and altered cell physiology. Autophagy is important in the clearance of toxic protein aggregates and damaged organelles, so under- or overactivity of this process often underlies the pathology of various neurodegenerative diseases. A well-characterized example is the involvement of autophagy in removing of SNCA in Parkinson disease (PD).108 A previous study showed that overexpression of RAB1 is able to rescue the neurodegenerative phenotype in PD models associated with SNCA accumulation.109 According to a recent observation, both SNCA overexpression and RAB1A-depletion result in increased accumulation of autophagic substrates and mislocalization of ATG9, suggesting the failure of autophagosome formation.91 In line with these findings, SNCA aggregation leads to Golgi fragmentation, a known symptom of PD, due to disruption of RAB1 homeostasis.110 Similarly, a protective role was revealed for Rab8A in the case of SNCA toxicity.111 Huntington disease (HD) is another well-known neurodegenerative disorder characterized by the accumulation of HTTcontaining protein aggregates. HTT was previously described as a GEF for RAB11.112 Several studies showed that RAB11 dysfunction or loss of its activity underlies the cellular pathomechanism of HD113-115 and in various HD models, RAB11 is able to rescue the neuronal loss.46,116 Besides RAB11, RAB8 is also involved in HD and its mislocalization results in perturbation of post-Golgi trafficking to lysosomes117 that may affect autophagic degradation. Dysregulation of the autophagic process is found in HD as well.118 However, the issue of whether inefficient autophagy is a cause or an implication of neurodegenerative diseases still remains open. Further investigation of the role of autophagic




the number of phagophores, indicated by ultrastructural studies, suggesting that this GTPase is required for the early steps of autophagy. The exact molecular mechanism of the contribution of RAB9 to autophagosome formation remains unknown, although this study suggested that it directly participates in providing a TGN-derived membrane source for phagophore expansion. The upstream effectors of RAB9 are poorly characterized; its only known GEF is DENND2A,99 but it has not yet been implicated in autophagy. RAB33A and RAB33B are Golgi-resident RAB proteins with a well-defined role in the retrograde transport between the Golgi and ER.100 Recently, it was described that expression of constitutively active RAB33B suppressed autophagy, whereas its depletion has no detectable effect on it.51 This study suggests that RAB33B has a role in autophagosome formation, likely through its interaction with ATG16L1. Consistent with this, the RAB33 GAP TBC1D25/OATL1 was identified as an LC3-binding protein.52 This study showed that OATL1 is recruited to the autophagosomes through its physical interaction with LC3. The GAP activity on RAB33B of this protein was found to be required for the proper fusion of autophagosomes with lysosomes. These results suggest a possible feedback loop in autophagy regulation by RAB33B, which is also involved in autophagosome maturation, since the overexpression of RAB33B or TBC1D25 inhibits the fusion of autophagosomes with lysosomes. However, the molecular mechanism by which RAB33B contributes to the regulation of autophagy is still unknown.

RABs in these disorders may provide a deeper insight into this question. Autophagy impairment has also been involved in neurodegeneration observed in lysosomal storage disorders, such as Niemann–Pick Type C disease, which is associated with cholesterol and sphingolipid accumulation in late endosomes and lysosomes.119,120 Interestingly, both RAB8121 and RAB9122,123 are able to rescue the defects in lipid trafficking in various Niemann– Pick Type C disease models.

Concluding Remarks The amount of data concerning the possible involvement of RAB proteins in the regulation of autophagy has grown almost exponentially during the past several years. These basic observations opened a new, promising and fascinating field of autophagy research. The results summarized above clearly show that a specific set of RAB small GTPases have a deep impact in the molecular mechanisms of the autophagic process (Fig. 5). RAB proteins implicated in autophagy can contribute to this process directly

and indirectly as well. RAB5 and RAB7 participate in several steps of the autophagic process: both of them take part in the upstream regulation of autophagy induction and in the formation of autophagosomes. RAB7 has further roles in the maturation and trafficking process of autophagosomes and amphisomes. Although RAB5, as a part of the BECN1-PIK3C3 complex, regulates directly autophagosome formation, the contribution of RAB7 to the maturation process of autophagosomes seems to be more indirect and dependent on its endosomal functions. RAB1 and RAB11 are undoubtedly among the key participants of the earliest steps of autophagosome formation through providing membrane from various sources for the expansion of the phagophore. On top of these, RAB1 has a well-characterized role in the recruitment of several ATG proteins and in the regulation of membrane transport toward the PAS. Furthermore, RAB11, through the regulation of the subcellular localization of Hook protein, may coordinate the maturation process of autophagic and endosomal pathways. Two Golgi-localized RABs, RAB9 and RAB33, are also implicated in autophagy. RAB9 has a well-defined role in autophagosome formation, although, the molecular mechanism

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Figure 5. The functions of RAB proteins in autophagy. The schematic picture shows the interactions of autophagy with the endocytic and secretory pathways. A set of RAB small GTPases play a role in the earliest steps of autophagosome formation by providing various membrane sources for the PAS. RAB5 also participates in this autophagic stage, through its interactions with the PIK3C3-BECN1 complex. RAB7 has both direct roles—in the transport of autophagosomes and amphisomes—and indirect roles in the fusion process of autophagosomes with late endosomes. Besides its roles in PAS formation, RAB11 regulates amphisome formation and coordinates the autophagic and endosomal pathways. ER, endoplasmic reticulum; TGN, trans-Golgi network; PAS, phagophore assembly site.

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able to interact with core components of the autophagic machinery and have an important role in orchestrating this degradative route.42,124 However, the work to date represents only the first steps in understanding the molecular basis of the dynamic interactions between the RAB cascades and core autophagic machinery. As several RABs are implicated in various diseases, identifying unknown regulators and mechanisms may establish the promising development of more specific drugs and therapeutic approaches. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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is as yet poorly understood. However, as a previous study suggested, it may have a direct effect on autophagy by providing a TGN-derived membrane source for phagophore expansion. RAB33B and its GAP, TBC1D25, are involved both in the early and late stages of autophagy, but their exact roles in these processes are still unknown. Some observations suggest that other RABs (RAB8, RAB24, and RAB32) might also be involved in the regulation of autophagy, but their contribution requires further investigation. In spite of numerous studies focused on the role of small GTPases in autophagy, the role of their GAPs and GEFs in autophagy remained unclear. Recently, several observations showed that previously unknown RAB regulators are

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Volume 10 Issue 7



33. Lin W-J, Yang CY, Li LL, Yi YH, Chen KW, Lin Y-C, Liu CC, Lin CH. Lysosomal targeting of phafin1 mediated by Rab7 induces autophagosome formation. Biochem Biophys Res Commun 2012; 417:3542; PMID:22115783; http://dx.doi.org/10.1016/j. bbrc.2011.11.043 34. Sakurai A, Maruyama F, Funao J, Nozawa T, Aikawa C, Okahashi N, Shintani S, Hamada S, Ooshima T, Nakagawa I. Specific behavior of intracellular Streptococcus pyogenes that has undergone autophagic degradation is associated with bacterial streptolysin O and host small G proteins Rab5 and Rab7. J Biol Chem 2010; 285:22666-75; PMID:20472552; http://dx.doi.org/10.1074/jbc.M109.100131 35. Yamaguchi H, Nakagawa I, Yamamoto A, Amano A, Noda T, Yoshimori T. An initial step of GAScontaining autophagosome-like vacuoles formation requires Rab7. PLoS Pathog 2009; 5:e1000670; PMID:19956673; http://dx.doi.org/10.1371/journal. ppat.1000670 36. Flinn RJ, Yan Y, Goswami S, Parker PJ, Backer JM. The late endosome is essential for mTORC1 signaling. Mol Biol Cell 2010; 21:833-41; PMID:20053679; http://dx.doi.org/10.1091/mbc.E09-09-0756 37. Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β. EMBO J 2011; 30:470111; PMID:22068051; http://dx.doi.org/10.1038/ emboj.2011.398 38. Pilli M, Arko-Mensah J, Ponpuak M, Roberts E, Master S, Mandell MA, Dupont N, Ornatowski W, Jiang S, Bradfute SB, et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 2012; 37:223-34; PMID:22921120; http://dx.doi. org/10.1016/j.immuni.2012.04.015 39. Nozawa T, Aikawa C, Goda A, Maruyama F, Hamada S, Nakagawa I. The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection. Cell Microbiol 2012; 14:1149-65; PMID:22452336; http://dx.doi. org/10.1111/j.1462-5822.2012.01792.x 40. Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T, Komatsu M, Otsu K, Tsujimoto Y, Shimizu S. Discovery of Atg5/Atg7independent alternative macroautophagy. Nature 2009; 461:654-8; PMID:19794493; http://dx.doi. org/10.1038/nature08455 41. Longatti A, Lamb CA, Razi M, Yoshimura S, Barr FA, Tooze SA. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J Cell Biol 2012; 197:659-75; PMID:22613832; http://dx.doi.org/10.1083/ jcb.201111079 42. Knævelsrud H, Søreng K, Raiborg C, Håberg K, Rasmuson F, Brech A, Liestøl K, Rusten TE, Stenmark H, Neufeld TP, et al. Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. J Cell Biol 2013; 202:331-49; PMID:23878278; http://dx.doi.org/10.1083/ jcb.201205129 43. Puri C, Renna M, Bento CF, Moreau K, Rubinsztein DC. Diverse autophagosome membrane sources coalesce in recycling endosomes. Cell 2013; 154:1285-99; PMID:24034251; http://dx.doi. org/10.1016/j.cell.2013.08.044 44. Fader CM, Sánchez D, Furlán M, Colombo MI. Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells. Traffic 2008; 9:230-50; PMID:17999726; http:// dx.doi.org/10.1111/j.1600-0854.2007.00677.x 45. Fader CM, Sánchez DG, Mestre MB, Colombo MI. TI-VAMP/VAMP7 and VAMP3/cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathways. Biochim Biophys Acta 2009; 1793:1901-16; PMID:19781582; http://dx.doi.org/10.1016/j.bbamcr.2009.09.011

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75. Díaz-Troya S, Pérez-Pérez ME, Florencio FJ, Crespo JL. The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy 2008; 4:851-65; PMID:18670193 76. Jewell JL, Russell RC, Guan K-L. Amino acid signalling upstream of mTOR. Nat Rev Mol Cell Biol 2013; 14:133-9; PMID:23361334; http://dx.doi. org/10.1038/nrm3522 77. Dwivedi M, Sung H, Shen H, Park B-J, Lee S. Disruption of endocytic pathway regulatory genes activates autophagy in C. elegans. Mol Cells 2011; 31:477-81; PMID:21618079; http://dx.doi. org/10.1007/s10059-011-1035-1 78. Otomo A, Kunita R, Suzuki-Utsunomiya K, Ikeda J-E, Hadano S. Defective relocalization of ALS2/ alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation. FEBS Lett 2011; 585:730-6; PMID:21300063; http://dx.doi. org/10.1016/j.febslet.2011.01.045 79. Moyer BD, Allan BB, Balch WE. Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering. Traffic 2001; 2:268-76; PMID:11285137; http://dx.doi. org/10.1034/j.1600-0854.2001.1o007.x 80. Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, Ktistakis NT. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008; 182:685701; PMID:18725538; http://dx.doi.org/10.1083/ jcb.200803137 81. Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 2009; 11:14337; PMID:19898463; http://dx.doi.org/10.1038/ ncb1991 82. Ylä-Anttila P, Vihinen H, Jokitalo E, Eskelinen E-L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 2009; 5:1180-5; PMID:19855179; http://dx.doi. org/10.4161/auto.5.8.10274 83. Itakura E, Mizushima N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 2010; 6:76476; PMID:20639694; http://dx.doi.org/10.4161/ auto.6.6.12709 84. Ragusa MJ, Stanley RE, Hurley JH. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell 2012; 151:1501-12; PMID:23219485; http://dx.doi.org/10.1016/j.cell.2012.11.028 85. Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F. An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J Cell Biol 2010; 190:1005-22; PMID:20855505; http://dx.doi.org/10.1083/ jcb.200912089 86. Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C, Ichikawa R, Kinjo M, Ohsumi Y. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 2012; 198:219-33; PMID:22826123; http://dx.doi.org/10.1083/jcb.201202061 87. Yorimitsu T, Klionsky DJ. Atg11 Links Cargo to the Vesicle-forming Machinery in the Cytoplasm to Vacuole Targeting Pathway. 2005; 16:1593–605 88. He C, Song H, Yorimitsu T, Monastyrska I, Yen W-L, Legakis JE, Klionsky DJ. Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. J Cell Biol 2006; 175:925-35; PMID:17178909; http://dx.doi. org/10.1083/jcb.200606084

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1166 Autophagy

Volume 10 Issue 7

Coevolution of RAC Small GTPases and their Regulators GEF Proteins.

RAB GTPases and RAB-interacting proteins and their role in the control of cognitive functions.

Small GTPases as regulators of cell division.

Small RAB GTPases Regulate Multiple Steps of Mitosis.

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP).

The Roles of Small GTPases in Osteoclast Biology.

Rab GTPases: principles learned from yeast.

Are There Rab GTPases in Archaea?

Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures.

Emerging roles of small GTPases in secondary cell wall development.

Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells.

Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension.

Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension.

Sequential recruitment of Rab GTPases during early stages of phagocytosis.

Identification of Dck1 and Lmo1 as upstream regulators of the small GTPase Rho5 in Saccharomyces cerevisiae.

Splicing Regulators and Their Roles in Cancer Biology and Therapy.

Rab GTPases.

Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.

Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection.

[Rab GTPases networks in membrane traffic in Saccharomyces cerevisiae].

Regulators of carcinogenesis: emerging roles beyond their primary functions.

Rab proteins: the key regulators of intracellular vesicle transport.

Analysis of 52 Rab GTPases from channel catfish and their involvement in immune responses after bacterial infections.

lysosomal pathways.