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Flippases transport lipids across the membrane bilayer to generate and maintain asymmetry. The human fungal pathogen Candida albicans has 5 flippases, including Drs2, which is critical for filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to the antifungal drug fluconazole and copper ions. We show here that such a flippase mutant also has an altered distribution of phosphatidylinositol 4-phosphate [PI(4)P] and ergosterol. Analyses of additional lipid transporters, i.e. the flippases Dnf1-3, and all the oxysterol binding protein (Osh) family lipid transfer proteins, i.e. Osh2-4 and Osh7, indicate that they are not critical for filamentous growth. However, deletion of Osh4 alone, which exchanges PI(4)P for sterol, in a drs2 mutant can bypass the requirement for this flippase in invasive filamentous growth. In addition, deletion of the lipid phosphatase Sac1, which dephosphorylates PI(4)P, in a drs2 mutant results in a synthetic growth defect, suggesting that Drs2 and Sac1 function in parallel pathways. Together, our results indicate that a balance between the activities of two putative lipid transporters regulates invasive filamentous growth, via PI(4)P. In contrast, deletion of OSH4 in drs2 does not restore growth on fluconazole, nor on papuamide A, a toxin that binds PS in the outer leaflet of the plasma membrane, suggesting that Drs2 has additional role(s) in plasma membrane organization, independent of Osh4. As we show that C. albicans Drs2 localizes to different structures, including the Spitzenkörper, we investigated if a specific localization of Drs2 is critical for different functions, using a synthetic physical interaction approach to restrict/stabilize Drs2 at the Spitzenkörper. Our results suggest that the localization of Drs2 at the plasma membrane is critical for C. albicans growth on fluconazole and papuamide A, but not for invasive filamentous growth.

International audience; As the majority of fungi are nonmotile, polarized growth in response to an external signal enables them to search for nutrients and mating partners, and hence is crucial for survival and proliferation. Although the mechanisms underlying polarization in response to external signals has commonalities with polarization during mitotic division, during budding, and fission growth, the importance of diverse feedback loops regulating external signal-mediated polarized growth is likely to be distinct and uniquely adapted to a dynamic environment. Here, we highlight recent advances in our understanding of the mechanisms that are crucial for polarity in response to external signals in fungi, with particular focus on the roles of membrane traffic, small GTPases, and lipids, as well as the interplay between cell shape and cell growth.

In a number of elongated cells, such as fungal hyphae, a vesicle cluster is observed at the growing tip. This cluster, called a Spitzenkorper, has been suggested to act as a vesicle supply center, yet analysis of its function is challenging, as a majority of components identified thus far are essential for growth. Here, we probe the function of the Spitzenkorper in the human fungal pathogen Candida albicans, using genetics and synthetic physical interactions (SPI). We show that the C. albicans Spitzenkorper is comprised principally of secretory vesicles. Mutant strains lacking the Spitzenkorper component myosin light chain 1 (Mlc1) or having a SPI between Mlc1 and either another Spitzenkorper component, the Rab GTPase Sec4, or prenylated green fluorescent protein (GFP), are viable and still exhibit a Spitzenkorper during filamentous growth. Strikingly, all of these mutants formed filaments with increased diameters and extension rates, indicating that Mlc1 negatively regulates myosin V, Myo2, activity. The results of our quantitative studies reveal a strong correlation between filament diameter and extension rate, which is consistent with the vesicle supply center model for fungal tip growth. Together, our results indicate that the Spitzenkorper protein Mlc1 is important for growth robustness and reveal a critical link between filament morphology and extension rate. IMPORTANCE Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In Candida albicans, as in many filamentous fungi, a cluster of vesicles, called a Spitzenkorper, is observed at the tip of growing hyphae that is thought to function as a vesicle supply center. A central prediction of the vesicle supply center model is that the filament diameter is proportional to the extension rate. Here, we show that mutants lacking the Spitzenkorper component myosin light chain 1 (Mlc1) or having synthetic physical interactions between Mlc1 and either another Spitzenkorper component or prenylated GFP, are defective in filamentous growth regulation, exhibiting a range of growth rates and sizes, with a strong correlation between diameter and extension rate. These results suggest that the Spitzenkorper is important for growth robustness and reveal a critical link between filament morphology and extension rate.

Background The initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion. Results In the present study, we have used PDMS micro-fabrication to probe the ability of filamentous C. albicans cells to penetrate and grow invasively in substrates of different stiffness. We show that there is a threshold for penetration that corresponds to a stiffness of ~ 200 kPa and that invasive growth within a stiff substrate is characterized by dramatic filament buckling, along with a stiffness-dependent decrease in extension rate. We observed a striking alteration in cell morphology, i.e., reduced cell compartment length and increased diameter during invasive growth, that is not due to depolarization of active Cdc42, but rather occurs at a substantial distance from the site of growth as a result of mechanical compression. Conclusions Our data reveal that in response to this compression, active Cdc42 levels are increased at the apex, whereas active Rho1 becomes depolarized, similar to that observed in membrane protrusions. Our results show that cell growth and morphology are altered during invasive growth, suggesting stiffness dictates the host cells that C. albicans can penetrate.

Across eukaryotes, Rho GTPases such as Rac and Cdc42 play important roles in establishing cell polarity, which is a key feature of cell growth. In mammals and filamentous fungi, Rac targets large protein complexes containing NADPH oxidases (NOX) that produce reactive oxygen species (ROS). In comparison, Rho GTPases of unicellular eukaryotes were believed to signal cell polarity without ROS, and it was unclear whether Rho GTPases were required for ROS production in these organisms. We document here the first example of Rho GTPase–mediated post-transcriptional control of ROS in a unicellular microbe. Specifically, Cdc42 is required for ROS production by the NOX Fre8 of the opportunistic fungal pathogen Candida albicans. During morphogenesis to a hyphal form, a filamentous growth state, C. albicans FRE8 mRNA is induced, which leads to a burst in ROS. Fre8-ROS is also induced during morphogenesis when FRE8 is driven by an ectopic promoter; hence, Fre8 ROS production is in addition controlled at the post-transcriptional level. Using fluorescently tagged Fre8, we observe that the majority of the protein is associated with the vacuolar system. Interestingly, much of Fre8 in the vacuolar system appears inactive, and Fre8-induced ROS is only produced at sites near the hyphal tip, where Cdc42 is also localized during morphogenesis. We observe that Cdc42 is necessary to activate Fre8-mediated ROS production during morphogenesis. Cdc42 regulation of Fre8 occurs without the large NOX protein complexes typical of higher eukaryotes and therefore represents a novel form of ROS control by Rho GTPases.

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth. Deletion of the Arf GTPase Arl1 results in hyphae that are shorter as well as reduced virulence. How Arl1 is regulated during hyphal growth, a process characteristic of filamentous fungi, yet absent in S. cerevisiae, is unknown. Here, we investigated the importance of the Rab6 homolog, Ypt6, in Arl1-dependent hyphal growth and determined that YPT6 overexpression specifically rescued the hyphal growth defect of an arl1 mutant, but not the converse. Furthermore, we show that deletion of ARL1 results in an alteration of the distribution of the Rab8 homolog, Sec4, in hyphal cells and that this defect is restored upon YPT6 overexpression.

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa , develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe , respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans . The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth. Deletion of the Arf GTPase Arl1 results in hyphae that are shorter as well as reduced virulence. How Arl1 is regulated during hyphal growth, a process characteristic of filamentous fungi, yet absent in S. cerevisiae, is unknown. Here, we investigated the importance of the Rab6 homolog, Ypt6, in Arl1-dependent hyphal growth and determined that YPT6 overexpression specifically rescued the hyphal growth defect of an arl1 mutant, but not the converse. Furthermore, we show that deletion of ARL1 results in an alteration of the distribution of the Rab8 homolog, Sec4, in hyphal cells and that this defect is restored upon YPT6 overexpression.

International audience; Rho G-proteins and phosphatidylinositol phosphates, which are important for exocytosis, endocytosis and cytoskeleton organization, are key regulators of polarized growth in a range of organisms. The aim of the present brief review is to highlight recent findings and their implications with respect to the functions and interplay between Rho G-proteins and phosphatidylinositol phosphates in highly polarized fungal filamentous growth.

Rho G-proteins are critical for polarized growth, yet little is known about the dynamics of their activation during fungal filamentous growth. We first investigated the roles of Rho1 and Rho2 during Candida albicans filamentous growth. Our results show that Rho1 is required for invasive filamentous growth and that Rho2 is not functionally redundant with Rho1. Using fluorescent reporters, we examined the dynamics of the active form of Rho1 and Cdc42 during initiation and maintenance of hyphal growth. Quantitative analyses indicated that the distribution, but not the level, of these active G-proteins is altered during initial polarization upon germ tube emergence. A comparison of the dynamics of these active G-proteins during budding and hyphal growth indicates that a higher concentration of active Cdc42 was recruited to the germ tube tip than to the bud tip. During hyphal elongation, active Cdc42 remained tightly restricted to the hyphal tip, whereas active Rho1 was broadly associated with the apex and subsequently recruited to the cell division site. Furthermore, our data suggest that phosphoinositide-bis-phosphates are critical to stabilize active Rho1 at the growth site. Together, our results point towards different regulation of Cdc42 and Rho1 activity during initiation and maintenance of filamentous growth.

Summary Regulation of Rho G-proteins is critical for cytoskeletal organization and cell morphology in all eukaryotes. In the human opportunistic pathogen Candida albicans, Rac1 and its activator Dck1, a member of the CED5, Dock180, myoblast city family of guanine nucleotide exchange factors, are required for the budding to filamentous transition during invasive growth. We show that Lmo1, a protein with similarity to human ELMO1, is necessary for invasive filamentous growth, similar to Rac1 and Dck1. Furthermore, Rac1, Dck1 and Lmo1 are required for cell wall integrity, as the deletion mutants are sensitive to cell wall perturbing agents, but not to oxidative or osmotic stresses. The region of Lmo1 encompassing the ELMO and PH-like domains is sufficient for its function. Both Rac1 and Dck1 can bind Lmo1. Overexpression of a number of protein kinases in the rac1, dck1 and lmo1 deletion mutants indicates that Rac1, Dck1 and Lmo1 function upstream of the mitogen-activated protein kinases Cek1 and Mkc1, linking invasive filamentous growth to cell wall integrity. We conclude that the requirement of ELMO/CED12 family members for Rac1 function is conserved from fungi to humans.

International audience; The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes. membrane traffic | filamentous growth | polarity | morphogenesis

The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion. To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus. Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm. In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin. The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed. The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+. These results suggest that loop 4-5 is near or part of the cation binding site. Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone. The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation.

Morphogenesis, such as the transition from budding to filamentous growth in Candida albicans, is a fundamental process that is of general interest both because of its cell biology and for its implication in host–pathogen interactions. Condition-specific transcriptome analyses and large-scale gene deletion or inactivation studies, together with forward and reverse genetic approaches, have uncovered a number of components involved in the regulation of the cellular reorganization that takes place in C. albicans during morphogenesis. This chapter will summarize the main components involved in cellular morphogenesis and provide an update of our knowledge of the regulatory steps that control the initiation and maintenance of polarized growth during C. albicans hyphal formation.

International audience; Cell shape is a critical determinant for function. The baker's yeast Saccharomyces cerevisiae changes shape in response to its environment, growing by budding in rich nutrients, forming invasive pseudohyphal filaments in nutrient poor conditions and pear shaped shmoos for growth towards a partner during mating. The human opportunistic pathogen Candida albicans can switch from budding to hyphal growth, in response to numerous environmental stimuli to colonize and invade its host. Hyphal growth, typical of filamentous fungi, is not observed in S. cerevisiae. A number of internal cues regulate when and where yeast cells break symmetry leading to polarized growth and ultimately distinct cell shapes. This review discusses how cells break symmetry using the yeast S. cerevisiae paradigm and how polarized growth is initiated and maintained to result in dramatic morphological changes during C. albicans hyphal growth.

Many Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. In Candida 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 expressing ras1-C287S were 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. The C. albicans autoregulatory 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.

Escherichia coli preprotein translocase, composed of the peripheral membrane protein SecA bound at the integral membrane domain SecY/E, has been isolated and functionally reconstituted [Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., & Wickner, W. (1990) Cell 62, 649-657]. It is not known whether this purified enzyme supports multiple turnover cycles and how its kinetics compare with translocase in inverted membrane vesicles. We now report a quantitative comparison of the translocation of the outer membrane protein A precursor (proOmpA) by purified preprotein translocase and by inner membrane vesicles. ProOmpA cross-linked to bovine pancreatic trypsin inhibitor was used for quantitative titration of the functional translocation sites. The rate of proOmpA translocation per active site in this purified system is 25% of that observed in inverted membrane vesicles. Each functional site can catalyze multiple cycles of precursor translocation. These results indicate that the purified preprotein translocase properly reconstitutes translocation.

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.

Rho G proteins and their regulators are critical for cytoskeleton organization and cell morphology in all eukaryotes. In the opportunistic pathogen Candida albicans, the Rho G proteins Cdc42 and Rac1 are required for the switch from budding to filamentous growth in response to different stimuli. We show that Dck1, a protein with homology to the Ced-5, Dock180, myoblast city family of guanine nucleotide exchange factors, is necessary for filamentous growth in solid media, similar to Rac1. Our results indicate that Dck1 and Rac1 do not function in the same pathway as the transcription factor Czf1, which is also required for embedded filamentous growth. The conserved catalytic region of Dck1 is required for such filamentous growth, and in vitro this region directly binds a Rac1 mutant, which mimics the nucleotide-free state. In vivo overexpression of a constitutively active Rac1 mutant, but not wild-type Rac1, in a dck1 deletion mutant restores filamentous growth. These results indicate that the Dock180 guanine nucleotide exchange factor homologue, Dck1 activates Rac1 during invasive filamentous growth. We conclude that specific exchange factors, together with the G proteins they activate, are required for morphological changes in response to different stimuli.

We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen Candida albicans . We identified and disrupted RAC1 and show here that, in contrast to CDC42 , it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in C. albicans development.

The Rho G protein Cdc42 and its exchange factor Cdc24 are required for hyphal growth of the human fungal pathogen Candida albicans . Previously, we reported that strains ectopically expressing Cdc24 or Cdc42 are unable to form hyphae in response to serum. Here we investigated the role of these two proteins in hyphal growth, using quantitative real-time PCR to measure induction of hypha-specific genes together with time lapse microscopy. Expression of the hypha-specific genes examined depends on the cyclic AMP-dependent protein kinase A pathway culminating in the Efg1 and Tec1 transcription factors. We show that strains with reduced levels of CDC24 or CDC42 transcripts induce hypha-specific genes yet cannot maintain their expression in response to serum. Furthermore, in serum these mutants form elongated buds compared to the wild type and mutant budding cells, as observed by time lapse microscopy. Using Cdc24 fused to green fluorescent protein, we also show that Cdc24 is recruited to and persists at the germ tube tip during hyphal growth. Altogether these data demonstrate that the Cdc24/Cdc42 GTPase module is required for maintenance of hyphal growth. In addition, overexpression studies indicate that specific levels of Cdc24 and Cdc42 are important for invasive hyphal growth. In response to serum, CDC24 transcript levels increase transiently in a Tec1-dependent fashion, as do the G-protein RHO3 and the Rho1 GTPase activating protein BEM2 transcript levels. These results suggest that a positive feedback loop between Cdc24 and Tec1 contributes to an increase in active Cdc42 at the tip of the germ tube which is important for hypha formation.

Candida albicans , the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3 . Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.

Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

An aspartic residue (Asp55) located in the putative transmembrane α-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-α-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na+-binding site and 2) that the cation and sugar binding sites may be overlapping.

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H + , Na + or Li + -sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar—carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of |3H|-melibiose transport or its accumulation in isolated membrane vesicles. The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was investigated by further analyzing the Na+ (H+ or Li+)-coupled facilitated diffusion reactions catalyzed by the carrier in de-energized membrane vesicles, with particular emphasis on the reaction of sugar exchange at equilibrium. It is first shown that melibiose exchange at equilibrium proceeds without concomitant movement of Na+, i.e. the coupled cation is kinetically occluded during the melibiose exchange reaction. These results provide further experimental support for the model of Na+ sugar co-transport of the physiological substrate melibiose previously suggested (Bassilana, M., Pourcher, T., and Leblanc, G. (1987) J. Biol. Chem. 262, 16865-16870) in which: 1) the mechanisms of co-substrate binding to (or release from) the carrier are ordered processes on both the outer (Na+ first, sugar last) and inner membrane surfaces (sugar first, Na+ last) and give rise to a mirror-type model; 2) release of Na+ from the carrier on the inner membrane surface is very slow and rate-limiting for carrier cycling but is fast on the opposite side, contributing to the asymmetrical functioning of the permease. On the other hand, analysis of the exchange of identical sugars (homologous exchange) and different sugar analogs (heterologous exchange) indicates that the overall rate of sugar exchange reaction coupled to Na+ or Li+ is limited by the rate of one (or more) partial step(s) associated with the inflow of co-substrates and most probably by the rate of sugar release into the intravesicular medium. It is proposed that the variability of the facilitated diffusion reactions catalyzed by the carrier in the presence of different coupled cations and/or sugar analogs reflects variations in the rate of co-substrate release from the carrier on the inner membrane surface.

The substrate binding reaction of the melibiose carrier was analyzed by studying [3H]p-nitrophenyl-alpha-D-galactopyranoside (Np alpha Gal) binding to de-energized membrane vesicles from Escherichia coli RA11 as a function of H+ and Na+ (or Li+) concentrations. The data indicate first that Na+ (or Li+) activates Np alpha Gal binding at all pH values tested between 5.5 and 7.5 and second that H+ inhibits the Na+ (or Li+)-dependent activating effect on Np alpha Gal binding. Similar conclusions were drawn for melibiose and methyl-1-thio-beta-D-galactoside binding activities. Unexpectedly, Np alpha Gal, melibiose, and methyl-1-thio-beta-D-galactoside binding activities are insensitive to a variety of SH reagents which completely block transport activity. Quantitative analysis of the effects of H+ and Na+ ions on the parameters of Np alpha Gal binding show that 1) the maximal number of binding sites is constant irrespective of the concentration of Na+ or Li+ in the range of pH between 6 and 7.5 and 2) the apparent dissociation constant for Np alpha Gal binding varies with both Na+ and H+ according to a relation described by a linear combination of the concentration of H+ and the reciprocal of Na+ concentration. These results can be accounted for by a model which assumes sequential binding of the cation and substrate in this order and competition between Na+ and H+ for a common cationic binding site on the porter. Predictions of the proposed binding model for a carrier mechanism catalyzing sugar transport according to a Na+ symport mode or a H+ symport mode are discussed.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.

The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained.

Bacterial permeases form a family of membrane proteins that actively transport nutrients according to a cation-solute cotransport mechanism. The purpose of this review is to consider the many perspectives offered by in vitro recombinant DNA and molecular biology techniques for analysis of structure-function relationships. In the first part of this review, the kinetic parameters that permit characterization of either the partial steps or the overall transport reaction are summarized. We then list the molecular properties (protein sequence and composition, secondary structure, biochemical identification) of the carriers readily available on cloning a structural gene into plasmids. Finally, the usefulness of site-specific, oligonucleotide-directed mutagenesis for investigation of the functional importance of given residues (cysteins and histidines) or domains of the transport proteins is illustrated.

Yeasts are unicellular fungi, which can be useful (bread, beer) or harmful to humans. Candida albicans is an opportunistic human pathogen, which can cause superficial as well as systemic infections, often life-threatening. One of the virulence factors of this organism is its ability to transit between an ovoid form and a filamentous form (hypha), capable of penetrating cellular tissues. During my thesis I studied the growth of C. albicans hyphae (diameter approximately 2 microns) in PDMS elastomers of various rigidity. I have observed that these hyphae are able to penetrate such environments with rigidities comparable to human tissues. My work was held into two parts.On one hand, I examined the penetration in cross-linked PDMS of a macroscopic one-millimeter diameter probe to better understand the mechanisms and forces involved, by analogy with the perforation of hyphae of microscopic size. I measured the force-strain relationships up to the point where the probe protrudes from the PDMS sample, which is a completely original aspect compared to the state of the art. I was able to show how the critical breaking force varied, but also the forces resistant to penetration up to the exit of the probe and to quantify the effects of the rigidity of the PMDS, but also of the speed.On the other hand, I worked with wild and mutated C. albicans strains. I developed and measured their growth in microfabricated PMDS membrane with chambers using real-time imaging. I first did a lot of work designing these membranes, most of the observations required a short working distance, which added a strong challenge on the manufacture of these membranes. The penetration of rigid media imposes on the filaments resistive stresses to their growth, as close as possible to the real situation. This makes the proposed approach an original one. Thanks to the results of the first part, we were able to determine the turgor pressure displayed in the hyphae using a recent growth model.; Les levures sont des champignons unicellulaires, qui peuvent être utiles (pain, bière) ou délétères pour l’homme. Candida albicans est une levure pathogène opportuniste de l’Homme, pouvant provoquer des infections superficielles mais aussi systémiques, pour lesquelles le pronostic vital est souvent engagé. Un des facteurs de virulence de cet organisme est sa capacité à transiter entre une forme ovoïde et une forme filamenteuse (hyphe), capable de pénétrer les tissus cellulaires. J’ai étudié pendant ma thèse la croissance d’hyphes de C. albicans (diamètre 2 microns environ) dans des élastomères de PDMS plus ou moins rigides. J’ai pu observer que ces hyphes sont capables de pénétrer de tels milieux aux rigidités comparables aux tissus humains. Mon travail s’est développé en deux parties. Dans une première partie, j’ai étudié la pénétration dans du PDMS réticulé d’une tige macroscopique millimétrique pour mieux comprendre les mécanismes et forces en jeu, par analogie avec la perforation des hyphes de taille microscopique. J’ai mesuré les relations forces-déformation jusqu’au point où la tige ressort de l’échantillon de PDMS, ce qui constitue un aspect complétement original par rapport à l’état de l’art. J’ai pu montrer comment variait la force critique de rupture, mais aussi les forces résistantes à la pénétration jusqu’à la sortie de la tige, et quantifier les effets de la rigidité du PMDS, mais aussi de la vitesse. Dans une deuxième partie, j’ai manipulé des souches de C. albicans sauvage et mutées. J’ai observé et mesuré leur croissance dans des dispositifs en PMDS microfabriqués à puits par imagerie en temps réel. J’ai d’abord réalisé un travail important de conception de ces dispositifs, sachant que la plupart des observations nécessitaient une distance de travail faible, ajoutant une contrainte forte sur la fabrication de ces dispositifs. La pénétration de milieux de rigidité et contrainte de rupture contrôlables imposent aux filaments des forces résistantes à leur croissance, au plus proche de la situation réelle, qui fait de l’approche proposée une démarche originale. Grâce aux résultats de la première partie, nous avons pu déterminer la pression de turgescence générée dans les hyphes en utilisant un modèle récent de croissance.