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

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

A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance

Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.     

Distribution and Functions of Sterols and Sphingolipids

Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.Typical examples of eukaryotic lipids, sterols, and sphingolipids can both be found in membranes from simple unicellular fungi and protists to multicellular animals and plants. Their versatile use as structural elements but also as signaling molecules has probably played an important role during the evolution of this large and diverse group of organisms. There are also many eukaryotes that have lost the ability to synthesize sterols de novo including nematodes, insects, and marine invertebrates, which have to take up sterols with their diet. Sterol biosynthesis has also been reported in a number of bacteria. Sphingolipids are more widely spread among prokaryotes than sterols and also show a greater variety of structures among the different eukaryotes.In this short review we will first give an overview about the diversity of sterol and sphingolipid structures and their distribution in nature. Then we will discuss their subcellular distribution. A brief technical section will add some information on the separation and detection of these lipid molecules. Subsequently, we will summarize different genetic approaches to study the functions of sterols and sphingolipids, and finally, we will discuss the functional and possible physical interactions of the two lipid classes within the cell. Far from being comprehensive, we will focus only on a few interesting aspects and try to give new view points, which are less frequently discussed.     

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.     

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.     

Microbial Eukaryotes that Lack Sterols

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen‐dependent multienzyme pathways. However, there are known exceptions―ciliated protozoans, such as Tetrahymena, along with diverse low‐oxygen‐adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol‐like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol‐like lipids in eukaryotes.     

Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking

The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking.     

Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium

The NPC1 family of proteins plays crucial roles in the intestinal absorption and intracellular trafficking of sterols. The Drosophila genome encodes two NPC1 homologs, one of which, NPC1a, is required for intracellular sterol trafficking in many tissues. Here we show that the other Drosophila NPC1 family member, NPC1b, is expressed in the midgut epithelium and that NPC1b is essential for growth during the early larval stages of development. NPC1b mutants are severely defective in sterol absorption, and the midgut epithelium of NPC1b mutants is deficient in sterols and sterol trafficking intermediates. By contrast, NPC1a mutants absorb sterols more efficiently than wild-type animals, and, unexpectedly, NPC1b;NPC1a double mutants absorb sterols as efficiently as wild-type animals. Together, these findings suggest that NPC1b plays an early role in sterol absorption, although sterol absorption continues at high efficiency through an NPC1a- and NPC1b-independent mechanism under conditions of impaired intracellular sterol trafficking.     

Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania . This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.     

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.     

Intracellular sterol dynamics

We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated.     

Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

溶酶体相关细胞器生物发生复合体相互作用组构成胞内体运输网络

随着高等生物中十几个新的参与囊泡运输的 Hermansky-Pudlak 综合征(HPS)蛋白质的发现, 认为可能存在一类新的囊泡运输通路。该通路主要由新近鉴定的 3 个被称为溶酶体相关细胞器生物发生复合体(BLOC)所组成, 被分别命名为BLOC-1、BLOC-2 和 BLOC-3。越来越多的证据表明这些复合体与以前认识较清楚的 AP3 和 HOPS 复合体共同在胞内体运输中起重要作用。这些复合体之间的相互作用构成了以胞内体和细胞骨架为连接纽带的参与蛋白质运输的复杂网络。该网络中的每个节点的相互作用可区分为复合体内和复合体外相互作用两大类。复合体之间的联系可以是来自不同复合体亚基间的直接相互作用, 也可以通过耦联的节点联结不同的复合体。解析这一复杂网络有助于进一步了解参与蛋白质和膜运输这一动态而精细网络的结构与功能。一旦该网络结构得到破坏, 则可能导致如 HPS 这类囊泡运输或细胞器发生障碍性疾病。     

Murine Hermansky-Pudlak syndrome genes: regulators of lysosome-related organelles

In the mouse, at least 16 genes regulate vesicle trafficking to specialized lysosome-related organelles, including platelet dense granules and melanosomes. Fourteen of these genes have been identified by positional cloning. All 16 mouse mutants are models for the genetically heterogeneous human disease, Hermansky-Pudlak Syndrome (HPS). Five HPS genes encode known vesicle trafficking proteins. Nine genes are novel, are found only in higher eukaryotes and encode members of three protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). Mutations in murine HPS genes, which encode protein co-members of BLOCs, produce essentially identical phenotypes. In addition to their well-known effects on pigmentation, platelet function and lysosome secretion, HPS genes control a wide range of physiological processes including immune recognition, neuronal functions and lung surfactant trafficking. Studies of the molecular functions of HPS proteins will reveal important details of vesicle trafficking and may lead to therapies for HPS.     

Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.     

Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.     

Glycolipid Trafficking in Drosophila Undergoes Pathway Switching in Response to Aberrant Cholesterol Levels

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons. The uptake mechanism and intracellular trafficking route of this simple glycolipid are largely conserved. Our principle finding is that cholesterol steers trafficking of the glycolipid between Golgi, lysosome, and recycling compartments. Our analyses support the idea that cholesterol storage in Drosophila triggers a switch in glycolipid trafficking from the biosynthetic to the degradative endolysosomal pathway, whereas cholesterol depletion eliminates recycling of the glycolipid. Unexpectedly, we observe a novel phenomenon we term “hijacking,” whereby lactosyl-ceramide diverts the trafficking pathway of an endocytic cargo, dextran, completely away from its lysosomal target. This work establishes that glycolipid trafficking in Drosophila undergoes changes similar to those seen in mammalian cells under conditions of cholesterol storage and therefore validates Drosophila as a suitable model organism in which to study lipid storage diseases.