Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

Newer Methods Drive Recent Insights into Rab GTPase Biology: An Overview.

The conserved Ypt/Rab GTPases regulate all major intracellular protein traffic pathways, including secretion, endocytosis and autophagy. These GTPases undergo distinct changes in conformation between their GTP- and GDP-bound forms and cycle between the cytoplasm and membranes with the aid of their upstream regulators. When activated on the membrane in the GTP-bound form, they recruit their downstream effectors, which include components of vesicular transport. Progress in the past 5 years regarding mechanisms of Rab action, functions, and the effects of disruption of these functions on the well-being of cells and organisms has been propelled by advances in methodologies in molecular and cellular biology. Here, we highlight methods used recently to analyze regulation, localization, interactions, and function of Rab GTPases and their roles in human disease. We discuss contributions of these methods to new insights into Rabs, as well as their future use in addressing open questions in the field of Rab biology.

Establishing Regulation of a Dynamic Process by Ypt/Rab GTPases : A Case for Cisternal Progression.

The prevailing model for transport within the Golgi is cisternal maturation. This process can be viewed as switching of cisternal markers using live-cell microscopy in yeast cells since the Golgi cisternae are separated, as opposed to the stacked Golgi seen in other organisms. It is also possible to determine which trafficking machinery components are required for this process by studying mutants depleted for these components. However, determining how cisternal maturation is regulated has been more challenging. The key for demonstrating regulation is not solely to stop the maturation when depleting a vesicular trafficking component, but also to illustrate a change in the speed. The obvious candidates for such regulation are the Ypt/Rab GTPases because of their established mode of action as regulators. Since the precise localization of the Golgi Ypts, Ypt1 and Ypt31, to specific Golgi cisternae has been controversial, we started by carefully colocalizing these Ypts with the Golgi cisternal markers using two independent approaches: immunofluorescence and live-cell microscopy. Next, the opposite effects of depletion versus constitutively activating Ypt mutations on Golgi morphology were determined. Finally, the ability of constitutively activating Ypt mutations to accelerate a specific cisternal-maturation step was established by live-cell time-lapse microscopy. Using these approaches, we defined three dynamic Golgi cisternae, early, intermediate, and late, separated two independent steps of cisternal maturation and showed their regulation by two different Ypts. Here, we discuss the major principles and precautions needed for each phase of this research, the main point being definition of a new criterion for regulation of a dynamic process versus requirement of a machinery structural component: acceleration of the dynamics.

Characterization of constitutive ER-phagy of excess membrane proteins.

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

TRAPPing a neurological disorder: from yeast to humans.

The modular complex TRAPP acts as an activator of a subgroup of Ypt/RAB GTPases. The substrate GTPases and TRAPP are conserved from yeast to human cells, required for secretion and macroautophagy/autophagy and implicated in human disease. All TRAPP complexes contain four core subunits essential for cell viability, and until recently there were no human diseases associated with any core TRAPP subunit. Recently, we reported a neurological disorder associated with a pathogenic variant of the core TRAPP subunit TRAPPC4. This variant results in lower levels of full-length TRAPPC4 protein and the TRAPP complex. A conditional mutation of the yeast homolog of TRAPPC4, Trs23, also results in a lower level of the protein and the TRAPP complex. Phenotypic analysis of the yeast mutant cells reveals a minor defect in secretion and a major defect in autophagy. Similarly, primary fibroblasts derived from human patients also exhibit minor and severe defects in secretion and autophagy, respectively. We propose that the autophagy defect caused by the pathogenic-TRAPPC4 variant results in the severe neurological disorder. Moreover, we hypothesize that low levels of the core TRAPP complex are more detrimental to autophagy than to secretion, and that the long-term autophagy defect is especially harmful to neuronal cells.Abbreviations: ER: endoplasmic reticulum; PM: plasma membrane; TRAPP: transport protein particle; Ypt: yeast protein transport.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

Ypt/Rab GTPases and their TRAPP GEFs at the Golgi.

The conserved Ypt/Rab GTPases regulate the different steps of all intracellular trafficking pathways. Ypt/Rabs are activated by their specific nucleotide exchangers termed GEFs, and when GTP bound, they recruit their downstream effectors, which mediate vesicular transport substeps. In the yeast exocytic pathway, Ypt1 and Ypt31/32 regulate traffic through the Golgi and the conserved modular TRAPP complex acts a GEF for both Ypt1 and Ypt31/32. However, the precise localization and function of these Ypts have been under debate, as is the identity of their corresponding GEFs. We have established that Ypt1 and Ypt31 reside on the two sides of the Golgi, early and late, respectively, and regulate Golgi cisternal progression. We and others have shown that whereas a single TRAPP complex, TRAPP II, activates Ypt31, three TRAPP complexes can activate Ypt1: TRAPPs I, III, and IV. We propose that TRAPP I and II activate Ypt1 and Ypt31, respectively, at the Golgi, whereas TRAPP III and IV activate Ypt1 in autophagy. Resolving these issues is important because both Rabs and TRAPPs are implicated in multiple human diseases, ranging from cancer to neurodegenerative diseases.

Autophagosome closure by ESCRT: Vps21/RAB5-regulated ESCRT recruitment via an Atg17-Snf7 interaction.

The macroautophagy/autophagy pathway includes successive steps of phagophore assembly structure formation, phagophore expansion, autophagosome (AP) closure and AP fusion with the lysosome/vacuole. Although information about regulators, factors and molecular mechanisms important for early and late steps of autophagy is abundant, information about AP closure is scarce. In 2017, we reported that the Vps21/RAB5 GTPase module regulates AP closure in yeast. In a recent paper, we show that Vps21 regulates the recruitment of ESCRT to APs to catalyze their closure by controlling an Atg17-Snf7 interaction. Thus, we identify a regulator, a factor, and a molecular mechanism important for AP closure. Abbreviations: AP: Autohagosome; Atg: autophagy-related gene; ESCRT: the endosomal sorting complex required for transport; ILVs: intralumenal vescicles.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

A Rab5 GTPase module is important for autophagosome closure.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Trs33-Containing TRAPP IV: A Novel Autophagy-Specific Ypt1 GEF.

Ypt/Rab GTPases, key regulators of intracellular trafficking pathways, are activated by guanine-nucleotide exchange factors (GEFs). Here, we identify a novel GEF complex, TRAPP IV, which regulates Ypt1-mediated autophagy. In the yeast Saccharomyces cerevisiae, Ypt1 GTPase is required for the initiation of secretion and autophagy, suggesting that it regulates these two distinct pathways. However, whether these pathways are coordinated by Ypt1 and by what mechanism is still unknown. TRAPP is a conserved modular complex that acts as a Ypt/Rab GEF. Two different TRAPP complexes, TRAPP I and the Trs85-containing TRAPP III, activate Ypt1 in the secretory and autophagic pathways, respectively. Importantly, whereas TRAPP I depletion copies Ypt1 deficiency in secretion, depletion of TRAPP III does not fully copy the autophagy phenotypes of autophagy-specific ypt1 mutations. If GEFs are required for Ypt/Rab function, this discrepancy implies the existence of an additional GEF that activates Ypt1 in autophagy. Trs33, a nonessential TRAPP subunit, was assigned to TRAPP I without functional evidence. We show that in the absence of Trs85, Trs33 is required for Ypt1-mediated autophagy and for the recruitment of core-TRAPP and Ypt1 to the preautophagosomal structure, which marks the onset of autophagy. In addition, Trs33 and Trs85 assemble into distinct TRAPP complexes, and we term the Trs33-containing autophagy-specific complex TRAPP IV. Because TRAPP I is required for Ypt1-mediated secretion, and either TRAPP III or TRAPP IV is required for Ypt1-mediated autophagy, we propose that pathway-specific GEFs activate Ypt1 in secretion and autophagy.

TRAPP Complexes in Secretion and Autophagy.

TRAPP is a highly conserved modular multi-subunit protein complex. Originally identified as a "transport protein particle" with a role in endoplasmic reticulum-to-Golgi transport, its multiple subunits and their conservation from yeast to humans were characterized in the late 1990s. TRAPP attracted attention when it was shown to act as a Ypt/Rab GTPase nucleotide exchanger, GEF, in the 2000s. Currently, three TRAPP complexes are known in yeast, I, II, and III, and they regulate two different intracellular trafficking pathways: secretion and autophagy. Core TRAPP contains four small subunits that self assemble to a stable complex, which has a GEF activity on Ypt1. Another small subunit, Trs20/Sedlin, is an adaptor required for the association of core TRAPP with larger subunits to form TRAPP II and TRAPP III. Whereas the molecular structure of the core TRAPP complex is resolved, the architecture of the larger TRAPP complexes, including their existence as dimers and multimers, is less clear. In addition to its Ypt/Rab GEF activity, and thereby an indirect role in vesicle tethering through Ypt/Rabs, a direct role for TRAPP as a vesicle tether has been suggested. This idea is based on TRAPP interactions with vesicle coat components. While much of the basic information about TRAPP complexes comes from yeast, mutations in TRAPP subunits were connected to human disease. In this review we will summarize new information about TRAPP complexes, highlight new insights about their function and discuss current controversies and future perspectives.

Regulation of Golgi Cisternal Progression by Ypt/Rab GTPases.

Current models entail that transport through the Golgi-the main sorting compartment of the cell-occurs via cisternal progression/maturation and that Ypt/Rab GTPases regulate this process. However, there is very limited evidence that cisternal progression is regulated, and no evidence for involvement of Ypt/Rab GTPases in such a regulation. Moreover, controversy about the placement of two of the founding members of the Ypt/Rab family, Ypt1 and Ypt31, to specific Golgi cisternae interferes with addressing this question in yeast, where cisternal progression has been extensively studied. Here, we establish the localization of Ypt1 and Ypt31 to opposite faces of the Golgi: early and late, respectively. Moreover, we show that they partially overlap on a transitional compartment. Finally, we determine that changes in Ypt1 and Ypt31 activity affect Golgi cisternal progression, early-to-transitional and transitional-to-late, respectively. These results show that Ypt/Rab GTPases regulate two separate steps of Golgi cisternal progression.

Rab11a Mediates Vascular Endothelial-Cadherin Recycling and Controls Endothelial Barrier Function.

OBJECTIVE: Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity. APPROACH AND RESULTS: Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis. CONCLUSIONS: Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.

A Role for Macro-ER-Phagy in ER Quality Control.

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20-50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.

Ypt1 and TRAPP interactions: optimization of multicolor bimolecular fluorescence complementation in yeast.

Ypt/Rab GTPases are conserved molecular switches that regulate the multiple vesicular transport steps of all intracellular trafficking pathways. They are stimulated by guanine-nucleotide exchange factors (GEFs). In yeast, Ypt1 regulates transport from the endoplasmic reticulum (ER) to two alternative pathways: secretion and autophagy. Ypt1 is activated by TRAPP, a modular multi-subunit GEF. Whereas TRAPP I activates Ypt1 to mediate transport through the Golgi, TRAPP III, which contains all the subunits of TRAPP I plus Trs85, activates Ypt1-mediated transport to autophagosomes. The functional pair Ypt31/32 regulates traffic in and out of the trans-Golgi and is activated by TRAPP II, which consists of TRAPP I plus two specific subunits, Trs120 and Trs130. To study the interaction of Ypts with specific TRAPP subunits and interactions between the different subunits of TRAPP, including the cellular sites of these interactions, we have employed a number of approaches. One approach that we have recently optimized for the use in yeast is multicolor bimolecular fluorescence complementation (BiFC). BiFC, which employs split fluorescent tags, has emerged as a powerful approach for determining protein interaction in vivo. Because proteins work in complexes, the ability to determine more than one interaction at a time using multicolor BiFC is even more powerful. Defining the sites of protein interaction is possible by co-localization of the BiFC puncta with compartmental markers. Here, we describe a set of plasmids for multicolor BiFC optimized for use in yeast. We combined their use with a set of available yeast strains that express red fluorescence compartmental markers. We have recently used these constructs to determine Ypt1 and TRAPP interactions in two different processes: intracellular trafficking and autophagy.

Ypt/Rab GTPases: principles learned from yeast.

Ypt/Rab GTPases are key regulators of all membrane trafficking events in eukaryotic cells. They act as molecular switches that attach to membranes via lipid tails to recruit their multiple downstream effectors, which mediate vesicular transport. Originally discovered in yeast as Ypts, they were later shown to be conserved from yeast to humans, where Rabs are relevant to a wide array of diseases. Major principles learned from our past studies in yeast are currently accepted in the Ypt/Rab field including: (i) Ypt/Rabs are not transport-step specific, but are rather compartment specific, (ii) stimulation by nucleotide exchangers, GEFs, is critical to their function, whereas GTP hydrolysis plays a role in their cycling between membranes and the cytoplasm for multiple rounds of action, (iii) they mediate diverse functions ranging from vesicle formation to vesicle fusion and (iv) they act in GTPase cascades to regulate intracellular trafficking pathways. Our recent studies on Ypt1 and Ypt31/Ypt32 and their modular GEF complex TRAPP raise three exciting novel paradigms for Ypt/Rab function: (a) coordination of vesicular transport substeps, (b) integration of individual transport steps into pathways and (c) coordination of different transport pathways. In addition to its amenability to genetic analysis, yeast provides a superior model system for future studies on the role of Ypt/Rabs in traffic coordination due to the smaller proteome that results in a simpler traffic grid. We propose that different types of coordination are important also in human cells for fine-tuning of intracellular trafficking, and that coordination defects could result in disease.

A Vps21 endocytic module regulates autophagy.

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome--endocytosis and autophagy--converge through the Vps21 and Ypt7 GTPase modules.