Lipid outward translocation by ABC proteins

In humans, about 50 ABC proteins play physiologically important roles. Many ABC proteins are involved in lipid outward translocation and lipid homeostasis in the body, and defects in their functions cause various diseases. However, the precise mechanisms of substrate transport remain unclear. In bacteria, several ABC proteins are involved in the transport of lipoproteins and lipopolysaccharides from the inner to outer membrane, and their functioning is a prerequisite for survival. Their functions can be divided into “flip-flop” and “projection”. In this review, human ABC proteins are compared to bacterial proteins to elucidate their mechanisms.     

Auxin transport: ABC proteins join the club

The isolation of potential auxin carriers from Arabidopsis thaliana marks a breakthrough in the characterization of elements involved in auxin delivery. Current models suggest that asymmetrical localization of auxin uptake and efflux carriers within the plasma membrane control the establishment of auxin gradients via facilitated transport. Now, the analysis of mutants defective in Arabidopsis ABC proteins indicates that primary active transport might participate in the control of auxin homeostasis as well.     

Ribonucleoparticle-independent transport of proteins into mammalian microsomes

There are at least two different mechanisms for the transport of secretory proteins into the mammalian endoplasmic reticulum. Both mechanisms depend on the presence of a signal peptide on the respective precursor protein and involve a signal peptide receptor on the cis-side and signal peptidase on the trans-side of the membrane. Furthermore, both mechanisms involve a membrane component with a cytoplasmically exposed sulfhydryl. The decisive feature of the precursor protein with respect to which of the two mechanisms is used is the chain length of the polypeptide. The critical size seems to be around 70 amino acid residues (including the signal peptide). The one mechanism is used by precursor proteins larger than about 70 amino acid residues and involves two cytosolic ribonucleoparticles and their receptors on the microsomal surface. The other one is used by small precursor proteins and relies on the mature part within the precursor molecule and a cytosolic ATPase.     

Lipid modification of proteins and their membrane transport

An effective method for artificial attachment of lipid anchors to water-soluble proteins has been developed. To this end, a protein molecule is modified in a system of reversed micelles by a water-insoluble reagent, e.g. fatty acid chloride. Fatty acylated proteins acquire an ability to translocate across lipid membranes and penetrate intact cells. This principle of imparting transmembrane properties to water-soluble proteins makes it possible to realize in vivo a direct transport of antibodies across the hemato-encephalic barrier into the brain and to develop a method for virus suppression by fatty acylated anti-viral antibodies capable of penetrating infected cells. The effect of a drastic increase in the biological activity of exogenous protein factors, e.g. Staphylococcus aureus enterotoxin A, as a result of their artificial fatty acylation has been discovered. The above-mentioned phenomena are discussed in relation to the in vivo data, indicating that post-translational modification of proteins by fatty acids and phospholipids is very widespread in nature and evidently plays an important role in protein transport and sorting. In this connection, lipid modification of proteins is regarded as a possible general step of protein transport in vivo.     

Functional analysis of candidate ABC transporter proteins for sitosterol transport

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.     

Function of prokaryotic and eukaryotic ABC proteins in lipid transport

ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry.     

Flavonoid transport by mammalian endothelial cells

Despite the ever-growing body of literature reporting the effects of flavonoids on animals at both the cellular and systemic levels, one of the most basic questions-"Are the effects of flavonoids on animal cells initiated through their interaction with extracellular targets or intracellular targets?"-has yet to be addressed. Because many effects of flavonoids on cells can be detected within minutes of flavonoid application and because flavonoids diffuse across lipid membranes slowly or not at all, intracellular mechanisms would necessitate a flavonoid transport system for rapid flavonoid uptake. The specific aims of this investigation were (1) to determine if endothelial cells contain a mechanism that mediates rapid flavonoid uptake and (2) to provide evidence for or against the hypothesis that rapid flavonoid effects on endothelial cell synthesis of prostacyclin and endothelin are initiated through the interaction of flavonoids with intracellular targets. Data show that bovine and human aortic endothelial cells possess a transport system that mediates rapid uptake of the flavonoid morin and suggest that the flavonoid uptake system utilizes a variety of oxygenated phenolic compounds as substrates. Further investigation into flavonoid transport should expedite future investigation into the mechanisms of flavonoid actions, because it may allow research to focus on the cellular locations where flavonoids are concentrated. Although endothelial cells contain a mechanism for the rapid uptake of morin, data reported herein suggest that morin initiates its rapid effects on endothelial cell synthesis of prostacyclin and endothelin through an interaction with extracellular targets.     

Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.

In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the "twilight zone," their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.     

ABC transporters in lipid transport

Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport. The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids. Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug. The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene. Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins. Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen. Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye. ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter. A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.     

ABC transporter-facilitated ATP conductive transport

The concept that the cystic fibrosis (CF) transmembraneconductance regulator, the protein product of the CF gene, can conduct larger multivalent anions such as ATP as well asCl is controversial. Inthis review, I examine briefly past findings that resulted incontroversy. It is not the goal of this review to revisit thesedisparate findings in detail. Rather, I focus intently on more recentstudies, current studies in progress, and possible future directionsthat arose from the controversy and that may reconcile this issue.Important questions and hypotheses are raised as to the physiologicalroles that ATP-binding cassette (ABC) transporter-facilitated ATPtransport and signaling may play in the control of epithelial cellfunction. Perhaps the identification of key biological paradigms forABC transporter-mediated extracellular nucleotide signaling may unifyand guide the CF research community and other research groupsinterested in ABC transporters toward understanding why ABCtransporters facilitate ATP transport.

     

Lipid transport proteins in malaria,from Plasmodium parasites to their hosts

Eukaryotic unicellular pathogens from the genus Plasmodium are the etiological agents of malaria, a disease that persists over a wide range of vertebrate species, including humans. During its dynamic lifecycle, survival in the different hosts depends on the parasite's ability to establish a suitable environmental milieu. To achieve this, specific host processes are exploited to support optimal growth, including extensive modifications to the infected host cell. These modifications include the formation of novel membranous structures, which are induced by the parasite. Consequently, to maintain a finely tuned and dynamic lipid environment, the organisation and distribution of lipids to different cell sites likely requires specialised lipid transfer proteins (LTPs). Indeed, several parasite and host-derived LTPs have been identified and shown to be essential at specific stages. Here we describe the roles of LTPs in parasite development and adaptation to its host including how the latest studies are profiting from the improved genetic, lipidomic and imaging toolkits available to study Plasmodium parasites. Lastly, a list of predicted Plasmodium LTPs is provided to encourage research in this field.     

Mutational analysis of ABC proteins

The 49 human members of the ATP-binding cassette (ABC) family of proteins are involved in a wide range of activities such as active transport of compounds across membranes, extraction of compounds out of membranes, functioning as ion channels, or regulators of channel activity. Mutations and/or overexpression of many of the proteins can have adverse effects on health. A goal in the study of ABC proteins is to understand their mechanisms of action. This review will focus on the mutational approaches that have been used to study the structure and mechanisms of some ABC proteins.     

Lipid transfer proteins

Translocations of various lipid species between membranes have been extensively studied. The transport of water-insoluble lipids is thought to require the participation of lipid transfer proteins (LTP). Several LTP, differing in their physiochemical properties and substrate specificities, have been purified to homogeneity from blood plasma, eucaryotic and procaryotic cells. Depending on their site of activity, they can be classified as extracellular and intracellular LTP. Extracellular LTP are found in the blood plasma and intracellular LTP, which were originally characterized as phospholipid exchange proteins, are ubiquitous in nature. Despite the enormous knowledge about their physicochemical properties and their function in vitro their physiological role has not been clearly demonstrated. However, their ubiquitous occurrence indicates an important role in cellular events. This review gives an overview of this interesting category of proteins, which are able to catalyze inter-membrane transfer and exchange of lipids.