Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.     

Determination of the membrane topology of Arv1 and the requirement of the ER luminal region for Arv1 function in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ARV1 encodes a 321 amino acid transmembrane protein localized to the endoplasmic reticulum (ER) and Golgi. It has been shown previously that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol biosyntheses, and may harbor sterol trafficking defects. Using C-terminal fusion to Suc2-His4, we determined the orientation of full-length Arv1 in the ER membrane. Once membrane topology was determined, we used this information and truncation analysis to establish the minimum protein length required for Arv1 function and phenotypic suppression. By understanding the topology of Arv1 we can now further analyze its putative lipid and glycosylphosphatidylinositol intermediate transport activities.     

Yeast cells lacking the ARV1 gene harbor defects in sphingolipid metabolism. Complementation by human ARV1

arv1Delta mutant cells have an altered sterol distribution within cell membranes (Tinkelenberg, A.H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M., and Sturley, S. L. (2000) J. Biol. Chem. 275, 40667-40670), and thus it has been suggested that Arv1p may be involved in the trafficking of sterol in the yeast Saccharomyces cerevisiae and also in humans. Here we present data showing that arv1Delta mutants also harbor defects in sphingolipid metabolism. [(3)H]inositol and [(3)H]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steady-state levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1Delta cells harbored defects in the rates of biosynthesis and steady-state levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1Delta cells. Moreover, these same studies demonstrated that arv1Delta cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1Delta cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1Delta cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1Delta cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.     

Saccharomyces cerevisiae Ste5 is important for induction and substrate specificity of Fus3 MAP kinase in the pheromone signaling pathway

The pheromone pathway is one of the mitogen activated protein kinase (MAPK) signaling pathways identified in Saccharomyces cerevisiae and is involved in both G1 cell cycle arrest and mating of cells. Fus3 functions at a branching point for G1 cell cycle arrest and mating responses in the signaling cascade, and the Fus3 MAPK uses components of both G1 arrest and mating routes as substrates. The Ste5 is a scaffold protein of the MAPK module and is essential for the activation of Fus3. However, it is not known how Ste5 is involved in the specific activation of Fus3 in G1 arrest and mating. In this study, we characterized several G1 arrest defective Ste5 mutants to better understand the roles of Ste5 in the regulation of Fus3. The level of Fus3 increased by treatment with alpha-factor. However, the alpha-factor effects were not readily apparent in the observation of yeast cells containing G1 arrest defective ste5 mutant. This suggests that Ste5 plays an essential role in Fus3 induction. Fus3 immune kinase assay of G1 arrest defective ste5 transformants revealed that Ste5 is important for substrate specificity of Fus3 for G1 arrest and/or mating.     

Role of Cdc42-Cla4 interaction in the pheromone response of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.     

Arv1 Regulates PM and ER Membrane Structure and Homeostasis But is Dispensable for Intracellular Sterol Transport

The pan‐eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild‐type and Arv1‐deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild‐type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C‐terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail‐anchored proteins involved in membrane homoeostasis .     

A link between very long chain fatty acid elongation and mating-specific yeast cell cycle arrest

Ceramides and sphingolipid intermediates are well-established regulators of the cell cycle. In the budding yeast Saccharomyces cerevisae, the complex sphingolipid backbone, ceramide, comprises a long chain sphingoid base, a polar head group, and a very long chain fatty acid (VLCFA). While ceramides and long chain bases have been extensively studied as to their roles in regulating cell cycle arrest under multiple conditions, the roles of VLCFAs are not well understood. Here, we used the yeast elo2 and elo3 mutants, which are unable to elongate fatty acids, as tools to explore if maintaining VLCFA elongation is necessary for cell cycle arrest in response to yeast mating. We found that both elo2 and elo3 cells had severely reduced mating efficiencies and were unable to form polarized shmoo projections that are necessary for cell-cell contact during mating. They also lacked functional MAP kinase signaling activity and were defective in initiating a cell cycle arrest in response to pheromone. Additional data suggests that mislocalization of the Ste5 scaffold in elo2 and elo3 mutants upon mating initiation may be responsible for the inability to initiate a cell cycle arrest. Moreover, the lack of proper Ste5 localization may be caused by the inability of mutant cells to mobilize PIP₂. We suggest that VLCFAs are required for Ste5 localization, which is a necessary event for initiating MAP kinase signaling and cell cycle arrest during yeast mating initiation.     

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

Counteractive control of polarized morphogenesis during mating by mitogen-activated protein kinase Fus3 and G1 cyclin-dependent kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.     

Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.     

p115 Rho GTPase activating protein interacts with MEKK1

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.     

Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.     

Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the hetero-trimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free Gβγ then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho^?) alleles of STE4: ste4-T320?A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

Persistent activation by constitutive Ste7 promotes Kss1-mediated invasive growth but fails to support Fus3-dependent mating in yeast

Mitogen-activated protein kinase kinase kinase-Ste11 (MAPKKK-Ste11), MAPKK-Ste7, and MAPK-Kss1 mediate pheromone-induced mating differentiation and nutrient-responsive invasive growth in Saccharomyces cerevisiae. The mating pathway also requires the scaffold-Ste5 and the additional MAPK-Fus3. One contribution to specificity in this system is thought to come from stimulus-dependent recruitment of the MAPK cascade to upstream activators that are unique to one or the other pathway. To test this premise, we asked if stimulus-independent signaling by constitutive Ste7 would lead to a loss of biological specificity. Instead, we found that constitutive Ste7 promotes invasion without supporting mating responses. This specificity occurs because constitutive Ste7 activates Kss1, but not Fus3, in vivo and promotes filamentation gene expression while suppressing mating gene expression. Differences in the ability of constitutive Ste7 variants to bind the MAPKs and Ste5 account for the selective activation of Kss1. These findings support the model that Fus3 activation in vivo requires binding to both Ste7 and the scaffold-Ste5 but that Kss1 activation is independent of Ste5. This scaffold-independent activation of Kss1 by constitutive Ste7 and the existence of mechanisms for pathway-specific promoter discrimination impose a unique developmental fate independently of any distinguishing external stimuli.     

Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.     

A Highlights from MBoC Selection: Combined computational and experimental analysis reveals mitogen-activated protein kinase–mediated feedback phosphorylation as a mechanism for signaling specificity

Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.