scholarly journals Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

2010 ◽  
Vol 9 (6) ◽  
pp. 960-970 ◽  
Author(s):  
Luiz R. Basso ◽  
Charles E. Gast ◽  
Yuxin Mao ◽  
Brian Wong
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Content Type ◽  
Empty Vector ◽  
Carbonyl Cyanide ◽  
Regulated Expression ◽  
Cytoplasmic Accumulation ◽  
Expression Plasmids

ABSTRACT A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Km s (expressed in micromolar concentrations) and V maxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

2010 ◽  
Vol 9 (10) ◽  
pp. 1455-1465 ◽  
Author(s):  
Laura A. Jones ◽  
Peter E. Sudbery
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Fluorescence Recovery After Photobleaching ◽  
Dynamic Properties ◽  
Secretory Vesicles ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Actin Cables ◽  
Cytochalasin A ◽  
Subapical Region

ABSTRACT During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

scholarly journals Dominant Negative Alleles ofSEC10Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

1998 ◽  
Vol 9 (7) ◽  
pp. 1725-1739 ◽  
Author(s):  
Dagmar Roth ◽  
Wei Guo ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Cycle Progression ◽  
Vesicle Docking ◽  
Amino Terminal ◽  
Exocyst Complex ◽  
Carboxy Terminal

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeastSaccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

scholarly journals The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome

2015 ◽  
Vol 83 (11) ◽  
pp. 4416-4426 ◽  
Author(s):  
Heather A. Danhof ◽  
Michael C. Lorenz
Keyword(s):  
Amino Acids ◽  
Candida Albicans ◽  
Hyphal Growth ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Ph Change ◽  
Content Type ◽  
Ammonia Release ◽  
Hyphal Formation

ABSTRACTCandida albicansis an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function.C. albicanscells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-geneATOfamily, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH changein vitroand in macrophages. When grown with amino acids as the sole carbon source, the deletion ofATO5or the expression of a dominant-negativeATO1G53Dallele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10ATOgenes accelerates ammonia release, and anato5Δ ATO1G53Ddouble mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allowC. albicansto overcome the macrophage innate immunity barrier.

Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

2012 ◽  
Vol 302 (11) ◽  
pp. E1419-E1430 ◽  
Author(s):  
Adelaide Del Viscovo ◽  
Agnese Secondo ◽  
Alba Esposito ◽  
Fernando Goglia ◽  
Maria Moreno ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
No Synthase ◽  
Dominant Negative ◽  
Gh3 Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Atp Binding Site ◽  
Carbonyl Cyanide ◽  
Intracellular Ca ◽  
Endogenous Nitric Oxide ◽  
Phosphatidylinositol 3

The role of 3,5,3′-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca2+ concentration ([Ca2+]i) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH3 cells in the absence or presence of extracellular Ca2+. When applied in Ca2+-free solution, T2 and T3 increased [Ca2+]i, in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by NG-nitro-l-arginine methyl ester and l- n5-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca2+]i increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca2+ stores did not interfere with the T2- and T3-induced [Ca2+]i increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca2+]i and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca2+]i increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na+/Ca2+ exchanger by CGP37157 significantly reduced the [Ca2+]i increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca2+]i and intracellular Na+ concentration; nimodipine reduced the [Ca2+]i increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na+/H+ pump by 5-( N-ethyl- N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na+/Ca2+ exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.

scholarly journals The Host GTPase Arf1 and Its Effectors AP1 and PICK1 Stimulate Actin Polymerization and Exocytosis To Promote Entry of Listeria monocytogenes

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Susan Saila ◽  
Gaurav Chandra Gyanwali ◽  
Mazhar Hussain ◽  
Antonella Gianfelice ◽  
Keith Ireton
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Surface Protein ◽  
Dominant Negative ◽  
Host Cells ◽  
Filamin A ◽  
Content Type ◽  
Bacterial Surface ◽  
Dominant Negative Allele

ABSTRACT Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.

scholarly journals The MARVEL Domain Protein Nce102 Regulates Actin Organization and Invasive Growth of Candida albicans

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Lois M. Douglas ◽  
Hong X. Wang ◽  
James B. Konopka
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Virulence Factors ◽  
Fungal Pathogen ◽  
Invasive Growth ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Actin Organization ◽  
Domain Protein

ABSTRACTInvasive growth of the fungal pathogenCandida albicansinto tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion ofNCE102did not cause the broad defects seen insur7Δcells. Instead, thence102Δmutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of abni1Δmutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. Thence102Δmutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surroundingC. albicansregulate morphogenesis and pathogenesis.IMPORTANCEThe plasma membrane promotes virulence of the human fungal pathogenCandida albicansby acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene,NCE102, caused a strong defect in formation of invasive hyphal growthin vitroand decreased virulence in mice. Thence102Δmutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

scholarly journals Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

2015 ◽  
Vol 83 (4) ◽  
pp. 1523-1535 ◽  
Author(s):  
Blessing Okai ◽  
Natalie Lyall ◽  
Neil A. R. Gow ◽  
Judith M. Bain ◽  
Lars-Peter Erwig
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Temporal Dynamics ◽  
Immune Defense ◽  
Dominant Negative ◽  
Phagosome Maturation ◽  
Hyphal Length ◽  
Content Type ◽  
Green Fluorescent

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogenCandida albicansundergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated byC. albicanshyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment ofC. albicanscells. Phagosomes containing liveC. albicanscells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages towardC. albicanscells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing byC. albicans. Therefore, Rab14 activity promotes phagosome maturation duringC. albicansinfection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

scholarly journals The Candida albicans Sur7 Protein Is Needed for Proper Synthesis of the Fibrillar Component of the Cell Wall That Confers Strength

2010 ◽  
Vol 10 (1) ◽  
pp. 72-80 ◽  
Author(s):  
Hong X. Wang ◽  
Lois M. Douglas ◽  
Vishukumar Aimanianda ◽  
Jean-Paul Latgé ◽  
James B. Konopka
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Wall Structure ◽  
Cell Wall Synthesis ◽  
Calcofluor White ◽  
Content Type ◽  
Membrane Compartment ◽  
Fibrillar Component ◽  
And Function

ABSTRACTTheCandida albicansplasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. Thesur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. Thesur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and β-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated thatsur7Δ cells contain decreased levels of β-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this,sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas β-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence β-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.

scholarly journals Roles of Ras1 Membrane Localization during Candida albicans Hyphal Growth and Farnesol Response

2011 ◽  
Vol 10 (11) ◽  
pp. 1473-1484 ◽  
Author(s):  
Amy E. Piispanen ◽  
Ophelie Bonnefoi ◽  
Sarah Carden ◽  
Aurelie Deveau ◽  
Martine Bassilana ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Hyphal Growth ◽  
Membrane Dynamics ◽  
Environmental Cues ◽  
Content Type ◽  
Lipid Modifications ◽  
Ras Gtpases

ABSTRACTMany Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. InCandida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressingras1-C287Swere delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. TheC. albicansautoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications.

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