Requirement of the N-terminal extension for vacuolar trafficking and transport activity of yeast Ycf1p,an ATP-binding cassette transporter

Ycf1p is the prototypical member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette (ABC) transporters. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent transport processes that result in resistance to cadmium and other xenobiotics. A feature common to many MRP proteins that distinguishes them from other ABC transporters is the presence of a hydrophobic N-terminal extension (NTE), whose function is not clearly established. The NTE contains a membrane spanning domain (MSD0) with five transmembrane spans and a cytosolic linker region (L0). The goal of this study was to determine the functional significance of the NTE of Ycf1p by examining the localization and functional properties of Ycf1p partial molecules, expressed either singly or together. We show that MSD0 plays a critical role in the vacuolar membrane trafficking of Ycf1p, whereas L0 is dispensable for localization. On the other hand, L0 is required for transport function, as determined by monitoring cadmium resistance. We also examine an unusual aspect of Ycf1p biology, namely, the posttranslational proteolytic processing that occurs within a lumenal loop of Ycf1p. Processing is shown to be Pep4p dependent and thus serves as a convenient marker for proper vacuolar localization. The processed fragments associate with each other, suggesting that these natural cleavage products contribute together to Ycf1p function.     

Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4^Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4^Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4.     

Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling

Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.     

Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.     

Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.     

alpha-Synuclein, oxidative stress and apoptosis from the perspective of a yeast model of Parkinson's disease

The neuronal protein α-synuclein (α-syn) has been suggested to be one of the factors linked to Parkinson's disease (PD). Several organisms, including the rat, mouse, worm, and fruit fly, are being used to study α-syn pathobiology. A new model organism was recently added to this armamentarium: the budding yeast Saccharomyces cerevisiae . The yeast system recapitulates many of the findings made with higher eukaryotes. For example, yeast cells expressing α-syn accumulate lipid droplets, have vacuolar/lysosomal defects, and exhibit markers of apoptosis, including the externalization of phosphatidylserine, the release of cytochrome c , and the accumulation of reactive oxygen species. This MiniReview focuses on the mechanisms by which α-syn induces oxidative stress and the mechanisms by which yeast cells respond to this stress. Three classes of therapeutics are discussed.     

Involvement of Rho-type GTPase in control of cell size in Saccharomyces cerevisiae

Maintaining specific cell size, which is important for many organisms, is achieved by coordinating cell growth and cell division. In the budding yeast Saccharomyces cerevisiae, the existence of two cell-size checkpoints is proposed: at the first checkpoint, cell size is monitored before budding at the G1/S transition, and at the second checkpoint, actin depolymerization occurring in the small bud is monitored before the G2/M transition. Morphological analyses have revealed that the small GTPase Rho1p participates in cell-size control at both the G1/S and the G2/M boundaries. One group of rho1 mutants (rho1A) underwent premature entry into mitosis, leading to the birth of abnormally small cells. In another group of rho1 mutants (rho1B), the mother cells failed to reach an appropriate size before budding, and expression of the G1 cyclin Cln2p began at an earlier phase of the cell cycle. Analyses of mutants defective in Rho1p effector proteins indicate that Skn7p, Fks1p and Mpk1p are involved in cell-size control. Thus, Rho1p and its downstream regulatory pathways are involved in controlling cell size in S. cerevisiae.     

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.     

Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.     

Functional domain analysis of the yeast ABC transporter Ycf1p by site-directed mutagenesis.

The yeast cadmium factor (Ycf1p) is a vacuolar protein involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors in yeast. It belongs to the superfamily of ATP binding cassette transporters, which includes the human cystic fibrosis transmembrane conductance regulator and the multidrug resistance-associated protein. To examine the functional significance of conserved amino acid residues in Ycf1p, we performed an extensive mutational analysis. Twenty-two single amino acid substitutions or deletions were generated by site-directed mutagenesis in the nucleotide binding domains, the proposed regulatory domain, and the fourth cytoplasmic loop. Mutants were analyzed phenotypically by measuring their ability to grow in the presence of Cd(2+). Expression and subcellular localization of the mutant proteins were examined by immunodetection in vacuolar membranes. For functional characterization of the Ycf1p variants, the kinetic parameters of glutathione S-conjugated leukotriene C(4) transport were measured. Our analysis shows that residues Ile(711), Leu(712), Phe(713), Glu(927), and Gly(1413) are essential for Ycf1p expression. Five other amino acids, Gly(663), Gly(756), Asp(777), Gly(1306), and Gly(1311), are critical for Ycf1p function, and two residues, Glu(709) and Asp(821), are unnecessary for Ycf1p biogenesis and function. We also identify several regulatory domain mutants in which Cd(2+) tolerance of the mutant strain and transport activity of the protein are dissociated.     

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.     

Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911

Yeast cadmium factor (Ycf1), an ATP-binding cassette (ABC) protein of the multidrug resistance protein subfamily, is a vacuolar GS-conjugate transporter required for heavy metal and drug detoxification. There is evidence that phosphorylation may play a critical role in the function of ABC transporters from higher organisms. In this work, the possibility of Ycf1 phosphorylation was examined using site-directed mutagenesis. We demonstrate that Ser⁹⁰⁸ and Thr⁹¹¹, within the regulatory domain (R domain), are functionally important for Ycf1 transport activity and likely sites for phosphorylation. Mutation of these residues to alanine severely impaired the Ycf1-dependent cadmium detoxification capacity and transport activity, while replacement by acidic residues (mimicking phosphorylation) significantly suppressed the cadmium resistance and transport defects. Both in vitro treatment of Ycf1 with alkaline phosphatase and changes in the electrophoretic mobility of the S908A, T911A and double mutant S908A/T911A proteins supported the conclusion that Ycf1 is a phosphoprotein. The screening of the yeast kinome identified four protein kinases affecting cadmium detoxification, but none of them was involved directly in the phosphorylation of Ycf1. Our data strongly implicate Ycf1 phosphorylation as a key determinant in cadmium resistance in yeast, a significant finding given that very little is known about phosphorylation of ABC transporters in yeast.     

Spatial regulation of the exocyst complex by Rho1 GTPase

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.     

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.     

Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [(3)H]estradiol-beta-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.     

ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily

Summary: Members of the ATP-binding cassette (ABC) transporter superfamily exist in bacteria, fungi, plants, and animals and play key roles in the efflux of xenobiotic compounds, physiological substrates, and toxic intracellular metabolites. Based on sequence relatedness, mammalian ABC proteins have been divided into seven subfamilies, ABC subfamily A (ABCA) to ABCG. This review focuses on recent advances in our understanding of ABC transporters in the model organism Saccharomyces cerevisiae. We propose a revised unified nomenclature for the six yeast ABC subfamilies to reflect the current mammalian designations ABCA to ABCG. In addition, we specifically review the well-studied yeast ABCC subfamily (formerly designated the MRP/CFTR subfamily), which includes six members (Ycf1p, Bpt1p, Ybt1p/Bat1p, Nft1p, Vmr1p, and Yor1p). We focus on Ycf1p, the best-characterized yeast ABCC transporter. Ycf1p is located in the vacuolar membrane in yeast and functions in a manner analogous to that of the human multidrug resistance-related protein (MRP1, also called ABCC1), mediating the transport of glutathione-conjugated toxic compounds. We review what is known about Ycf1p substrates, trafficking, processing, posttranslational modifications, regulation, and interactors. Finally, we discuss a powerful new yeast two-hybrid technology called integrated membrane yeast two-hybrid (iMYTH) technology, which was designed to identify interactors of membrane proteins. iMYTH technology has successfully identified novel interactors of Ycf1p and promises to be an invaluable tool in future efforts to comprehensively define the yeast ABC interactome.     

Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.     

Selenodiglutathione uptake by the Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p

The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATP-dependent transport of glutathione-metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol36, 679-687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated. Using secretory vesicles isolated from a sec6-4 mutant transformed either with the plasmid harbouring YCF1 or the control plasmid, we establish that the glutathione-conjugate selenodigluthatione is a high-affinity substrate of this ATP-binding cassette transporter and that oxidized glutathione is also efficiently transported. Finally, we show that the presence of Ycf1p impairs the glutathione/oxidized glutathione ratio of cells subjected to a selenite stress. Possible mechanisms by which Ycf1p-mediated vacuolar uptake of selenodiglutathione and oxidized glutathione enhances selenite toxicity are discussed.