Ypt/Rab GTPases: Principles learned from yeast

Abstract

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.     

Trs130 Participates in Autophagy Through GTPases Ypt31/32 in Saccharomyces cerevisiae

Trs130 is a specific component of the transport protein particle II complex, which functions as a guanine nucleotide exchange factor (GEF) for Rab GTPases Ypt31/32. Ypt31/32 is known to be involved in autophagy, although the precise mechanism has not been thoroughly studied. In this study, we investigated the potential involvement of Trs130 in autophagy and found that both the cytoplasm‐to‐vacuole targeting (Cvt) pathway and starvation‐induced autophagy were defective in a trs130ts (trs130 temperature‐sensitive) mutant. Mutant cells could not transport Atg8 and Atg9 to the pre‐autophagosomal structure/phagophore assembly site (PAS) properly, resulting in multiple Atg8 dots and Atg9 dots dispersed in the cytoplasm. Some dots were trapped in the trans‐Golgi. Genetic studies showed that the effect of the Trs130 mutation was downstream of Atg5 and upstream of Atg1, Atg13, Atg9 and Atg14 on the autophagic pathway. Furthermore, overexpression of Ypt31 or Ypt32, but not of Ypt1, rescued autophagy defects in trs130ts and trs65ts (Trs130‐HA Trs120‐myc trs65Δ) mutants. Our data provide mechanistic insight into how Trs130 participates in autophagy and suggest that vesicular trafficking regulated by GTPases/GEFs is important in the transport of autophagy proteins from the trans‐Golgi to the PAS.     

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab‐A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP‐bound) versus wild‐type or constitutively active (GTP‐bound) RAB‐A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab‐A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB‐A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB‐A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF?Rab interactions will be crucial to unravel the co‐ordination of plant membrane traffic.     

Ypt and Rab GTPases: insight into functions through novel interactions.

Ypt/Rab GTPases are key regulators of vesicular transport in eukaryotic cells. During the past two years, a number of new Ypt/Rab-interacting proteins have been identified and shown to serve as either upstream regulators or downstream effectors. Proteins that interact with these regulators and effectors of Ypt/Rabs have also been identified, and together they provide new insights into Ypt/Rab mechanisms of action. The picture that emerges from these studies suggests that Ypt/Rabs function in multiple and diverse aspects of vesicular transport. In addition, not only are Ypt/Rabs highly conserved, but their functions and interactions are as well. Interestingly, crosstalk among Ypt/Rabs and between Ypt/Rabs and other signaling factors, suggest the possibility of coordination of the individual vesicular transport steps and of the protein transport machinery with other cellular processes.     

TRAPP complexes in membrane traffic: convergence through a common Rab

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.     

The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes

The multimeric membrane-tethering complexes TRAPPI and TRAPPII share seven subunits, of which four (Bet3p, Bet5p, Trs23p, and Trs31p) are minimally needed to activate the Rab GTPase Ypt1p in an event preceding membrane fusion. Here, we present the structure of a heteropentameric TRAPPI assembly complexed with Ypt1p. We propose that TRAPPI facilitates nucleotide exchange primarily by stabilizing the nucleotide-binding pocket of Ypt1p in an open, solvent-accessible form. Bet3p, Bet5p, and Trs23p interact directly with Ypt1p to stabilize this form, while the C terminus of Bet3p invades the pocket to participate in its remodeling. The Trs31p subunit does not interact directly with the GTPase but allosterically regulates the TRAPPI interface with Ypt1p. Our findings imply that TRAPPII activates Ypt1p by an identical mechanism. This view of a multimeric membrane-tethering assembly complexed with a Rab provides a framework for understanding events preceding membrane fusion at the molecular level.     

Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling

Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.     

The Ypt/Rab family and the evolution of trafficking in fungi

The evolution of the eukaryotic endomembrane system and the transport pathways of their vesicular intermediates are poorly understood. A common set of organelles and pathways seems to be present in all free-living eukaryotes, but different branches of the tree of life have a variety of diverse, specialized organelles. Rab/Ypt proteins are small guanosine triphosphatases with tissue-specific and organelle-specific localization that emerged as markers for organelle diversity. Here, I characterize the Rab/Ypt family in the kingdom Fungi, a sister kingdom of Animals. I identify and annotate these proteins in 26 genomes representing near one billion years of evolution, multiple lifestyles and cellular types. Surprisingly, the minimal set of Rab/Ypt present in fungi is similar to, perhaps smaller than, the predicted eukaryotic ancestral set. This suggests that the saprophytic fungal lifestyle, multicellularity as well as the highly polarized secretion associated with hyphal growth did not require any major innovation in the molecular machinery that regulates protein trafficking. The Rab/Ypt and other protein traffic-related families are kept small, not paralleling increases in genome size, in contrast to the expansion of such components observed in other branches of the tree of life, such as the animal and plant kingdoms. This analysis suggests that multicellularity and cellular diversity in fungi followed different routes from those followed by plants and metazoa.     

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.     

Activation of Rho and Rab GTPases dissociates Brucella abortus internalization from intracellular trafficking

Brucella abortus is an intracellular pathogen that relies on unconventional virulence factors to infect hosts. In non-professional phagocytes, Rho GTPases-activation by the Escherichia coli cytotoxic necrotizing factor (CNF) promoted massive Brucella entrance by membrane ruffling, a mechanism that differs from the common mode of entrance used by this bacterium in non-treated cells. Cytotoxic necrotizing factor treatment, however, did not alter the intracellular route followed by the wild type or non-virulent defined mutants. In contrast, expression of a constitutively active Rab5Q79L GTPase did not alter cell-invasion by Brucella but hampered its ability to reach the endoplasmic reticulum. The CNF-induced Brucella super-infection did not reduce the ability of host cells to synthesize DNA and progress through the cell cycle. Furthermore, CNF-treatment increased the isolation of Brucella-containing compartments by a factor of 15. These results demonstrate that in non-professional phagocytic cells, Brucella manipulates two different sets of GTPases during its biogenesis, being internalization and intracellular trafficking two consecutive but independent processes. Besides, CNF-induced super-infection demonstrates that Brucella does not interfere with crucial cellular processes and has shown its potential as tool to characterize the intracellular compartments occupied by this bacterium.     

Regulation of TORC2 function and localization by Rab5 GTPases in Saccharomyces cerevisiae

The evolutionarily conserved Target of Rapamycin (TOR) complex-2 (TORC2) is an essential regulator of plasma membrane homeostasis in budding yeast (Saccharomyces cerevisiae). In this yeast, TORC2 phosphorylates and activates the effector protein kinase Ypk1 and its paralog Ypk2. These protein kinases, in turn, carry out all the crucial functions of TORC2 by phosphorylating and thereby controlling the activity of at least a dozen downstream substrates. A previously uncharacterized interplay between the Rab5 GTPases and TORC2 signaling was uncovered through analysis of a newly suspected Ypk1 target. Muk1, one of two guanine nucleotide exchange factors for the Rab5 GTPases, was found to be a physiologically relevant Ypk1 substrate; and, genetic analysis indicates that Ypk1-mediated phosphorylation activates the guanine nucleotide exchange activity of Muk1. Second, it was demonstrated both in vivo and in vitro that the GTP-bound state of the Rab5 GTPase Vps21/Ypt51 physically associates with TORC2 and acts as a direct positive effector required for full TORC2 activity. These interrelationships provide a self-reinforcing control circuit for sustained up-regulation of TORC2-Ypk1 signaling. In this overview, we summarize the experimental basis of these findings, their implications, and speculate as to the molecular basis for Rab5-mediated TORC2 activation.     

GTPases in intracellular trafficking: an overview

Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.     

The role of Trs65 in the Ypt/Rab guanine nucleotide exchange factor function of the TRAPP II complex

The conserved modular complex TRAPP is a guanine nucleotide exchanger (GEF) for the yeast Golgi Ypt-GTPase gatekeepers. TRAPP I and TRAPP II share seven subunits and act as GEFs for Ypt1 and Ypt31/32, respectively, which in turn regulate transport into and out of the Golgi. Trs65/Kre11 is one of three TRAPP II-specific subunits. Unlike the other two subunits, Trs120 and Trs130, Trs65 is not essential for viability, is conserved only among some fungi, and its contribution to TRAPP II function is unclear. Here, we provide genetic, biochemical, and cellular evidence for the role of Trs65 in TRAPP II function. First, like Trs130, Trs65 localizes to the trans-Golgi. Second, TRS65 interacts genetically with TRS120 and TRS130. Third, Trs65 interacts physically with Trs120 and Trs130. Finally, trs65 mutant cells have low levels of Trs130 protein, and they are defective in the GEF activity of TRAPP II and the intracellular distribution of Ypt1 and Ypt31/32. Together, these results show that Trs65 plays a role in the Ypt GEF activity of TRAPP II in concert with the two other TRAPP II-specific subunits. Elucidation of the role played by Trs65 in intracellular trafficking is important for understanding how this process is coordinated with two other processes in which Trs65 is implicated: cell wall biogenesis and stress response.     

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85-91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.     

Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking

Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.     

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.     

Saccharomyces cerevisiae Rab-GDI displacement factor ortholog Yip3p forms distinct complexes with the Ypt1 Rab GTPase and the reticulon Rtn1p

Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Delta or rtn1Delta mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.     

Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.