Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.     

N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.     

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.     

Roles of Ras1 membrane localization during Candida albicans hyphal growth and farnesol response

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.     

H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae

Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine. Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane. The mechanism(s) by which this occurs is not well understood. In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus. However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p. Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment. The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318. An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S. Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol. Chem. 277:41268-41273, 2002). Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box. These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.     

Akr1p-dependent palmitoylation of Yck2p yeast casein kinase 1 is necessary and sufficient for plasma membrane targeting

The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membranes via palmitoylation of its C terminus. We have demonstrated that Yck2p traffics to the plasma membrane on secretory vesicles. Because Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, it is likely that Yck2p first associates with Golgi membranes, and then is somehow recruited to budding plasma membrane-destined vesicles. We show here that residues 499-546 are sufficient for minimal Yck2p palmitoylation and plasma membrane localization. We previously described normal plasma membrane targeting of a Yck2p construct with the final five amino acids of Ras2p substituting for the final two Cys residues of Yck2p. This Yck2p variant no longer requires Akr1p for membrane association, but targets normally. We have generated the C-terminal deletions previously shown to affect Yck2p membrane association in this variant to determine which residues are important for targeting and/or modification. We find that all of the sequences previously identified as important for plasma membrane association are required only for Akr1p-dependent modification. Furthermore, palmitoylation is sufficient for specific association of Yck2p with secretory vesicles destined for the plasma membrane. Finally, both C-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association.     

Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.     

Protein lipidation

Proteins are covalently modified with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins. The focus of this review is S-palmitoylation, the reversible addition of palmitate and other long-chain fatty acids to proteins at cysteine residues in a variety of sequence contexts. The functional consequences of palmitoylation are diverse. Palmitoylation facilitates the association of proteins with membranes, mediates protein trafficking, and more recently has been appreciated as a regulator of protein stability. Members of a family of integral membrane proteins that harbor a DHHC cysteine-rich domain mediate most cellular palmitoylation events. Here we focus on DHHC proteins that modify Ras proteins in yeast and mammalian cells.     

Probing protein palmitoylation at the yeast vacuole

A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles.     

Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.     

Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae

Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.     

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.     

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include – targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites – Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia – have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.     

Vac8p, a Vacuolar Protein with Armadillo Repeats, Functions in both Vacuole Inheritance and Protein Targeting from the Cytoplasm to Vacuole

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein–protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to β-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.