Lipid microdomains,lipid translocation and the organization of intracellular membrane transport (Review)

Eukaryotic cells contain hundreds of different lipid species that are not uniformly distributed among their membranes. For example, sphingolipids and sterols form gradients along the secretory pathway with the highest levels in the plasma membrane and the lowest in the endoplasmic reticulum. Moreover, lipids in late secretory organelles display asymmetric transbilayer arrangements with the aminophospholipids concentrated in the cytoplasmic leaflet. This lipid heterogeneity can be viewed as a manifestation of the fact that cells exploit the structural diversity of lipids in organizing intracellular membrane transport. Lipid immiscibility and the generation of phase-separated lipid domains provide a molecular basis for sorting membrane proteins into specific vesicular pathways. At the same time, energy-driven aminophospholipid transporters participate in membrane deformation during vesicle biogenesis. This review will focus on how selective membrane transport relies on a dynamic interplay between membrane lipids and proteins.     

Vesicular and non-vesicular lipid export from the ER to the secretory pathway

The endoplasmic reticulum is the site of synthesis of most glycerophospholipids, neutral lipids and the initial steps of sphingolipid biosynthesis of the secretory pathway. After synthesis, these lipids are distributed within the cells to create and maintain the specific compositions of the other secretory organelles. This represents a formidable challenge, particularly while there is a simultaneous and quantitatively important flux of membrane components stemming from the vesicular traffic of proteins through the pathway, which can also vary depending on the cell type and status. To meet this challenge cells have developed an intricate system of interorganellar contacts and lipid transport proteins, functioning in non-vesicular lipid transport, which are able to ensure membrane lipid homeostasis even in the absence of membrane trafficking. Nevertheless, under normal conditions, lipids are transported in cells by both vesicular and non-vesicular mechanisms. In this review we will discuss the mechanism and roles of vesicular and non-vesicular transport of lipids from the ER to other organelles of the secretory pathway.     

Sec14 related proteins in yeast

Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (Sec fourteen homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking.     

Multiple lipid transport pathways to the plasma membrane in yeast

The plasma membrane of the yeast Saccharomyces cerevisiae is devoid of lipid-synthesizing enzymes, but contains all classes of bilayer-forming lipids. As the lipid composition of the plasma membrane does not match any of the intracellular membranes, specific trafficking of lipids from internal membranes, especially the endoplasmic reticulum and the Golgi, to the cell periphery is required. Although the secretory pathway is an obvious route to translocate glycerophospholipids, sphingolipids and sterols to the plasma membrane, experimental evidence for the role of this pathway in lipid transport is rare. Addressing this issue in a systematic way, we labeled temperature-sensitive secretory yeast mutants (sec mutants) with appropriate lipid precursors, isolated the plasma membranes at high purity and quantified labeled lipids of this compartment. Shifting sec mutants to the restrictive temperature reduced transport of both proteins and lipids to the plasma membrane, indicating that the latter compounds are also trafficked to the cell periphery through the protein secretory pathway. However, efficient sec blocks did not abrogate protein and lipid transport, suggesting that parallel pathway(s) for the translocation of membrane components to the plasma membrane of yeast must exist.     

Membrane lipids and transporter function

Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins.     

Lipid-transfer proteins in biosynthetic pathways

Compartmentalization is a defining feature of eukaryotic cells that allows the spatial segregation of different functions, such as protein and lipid synthesis, and ensures their fidelity and efficiency. This imposes the need for an intense flux of metabolic intermediates between segregated enzymatic activities, as seen for the sequential transport of neosynthesized proteins through the segments of the secretory pathway during their post-translational modification. For lipid synthesis, the identification of proteins that transfer lipids between membranes has revealed an additional mechanism for this intercompartment exchange. The intense interest elicited by the lipid-transfer proteins over the last few years has led to the definition of their central role in key processes, such as lipid metabolism, membrane trafficking, and signaling.     

Impact of sphingolipids on protein membrane trafficking

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.     

ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

Proteins involved in lipid translocation in eukaryotic cells

Since the first discovery of ATP-dependent translocation of lipids in the human erythrocyte membrane in 1984, there has been much evidence of the existence of various ATPases translocating lipids in eukaryotic cell membranes. They include P-type ATPases involved in inwards lipid transport from the exoplasmic leaflet to the cytosolic leaflet and ABC proteins involved in outwards transport. There are also ATP-independent proteins that catalyze the passage of lipids in both directions. Five P-type ATPase involved in lipid transport have been genetically characterized in yeast cells, suggesting a pool of several proteins with partially redundant activities responsible for the regulation of lipid asymmetry. However, expression and purification of individual yeast proteins is still insufficient to allow reconstitution experiments in liposomes. In this review, we want to give an overview over current investigation efforts about the identification and purification of proteins that may be involved in lipid translocation.     

Processing of VSVG protein is not a rate-limiting step for its efflux from the Golgi complex

The secretory membrane system is comprised of membrane-bound organelles defined by specific sets of proteins that function in sequential modification of cargo proteins and lipids. This processing of cargo proteins and lipids is coupled to their secretory transport. Here, we investigated the effect of inhibiting N-glycan processing by swainsonine, an inhibitor of Golgi alpha1,2-mannosidase-II, on secretory transport of the thermo-reversible tsO45 mutant of vesicular stomatitis virus glycoprotein tagged with green fluorescent protein (VSVG-FP). Quantitative analysis using kinetic modeling combined with live cell imaging was used to derive the rate coefficients that delineate secretory transport of VSVG-FP. We found that neither inhibition of N-glycan processing nor elimination by mutagenesis of the first of the two asparagine-linked glycans had any significant effect on the rate of VSVG-FP transport through the Golgi. These data suggest that at least for VSVG, the multi-enzymatic process of N-glycan modification does not comprise a rate-limiting step for its Golgi efflux.     

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.     

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

The role of lipids in the biogenesis of integral membrane proteins

Most integral membrane proteins are cotranslationally inserted into the lipid bilayer. In prokaryotes, membrane insertion of the nascent chain takes place at the plasma membrane, whereas in eukaryotes insertion takes place into the endoplasmatic reticulum. In both kingdoms of life, however, the same membrane that acquaints the newly born membrane protein also synthesizes the bilayer lipids and thus ensures the balanced growth of the membrane as a whole. Recent evidence indicates that the lipid composition of the host membrane can determine the fate of the newborn membrane protein, as it can affect (1) the efficiency of translocation, (2) the topology of the resulting membrane protein, (3) its stability, (4) its assembly into oligomeric complexes, (5) its transport and sorting along the secretory pathway, and (6) its enzymatic activity. The lipid composition of the membrane thus can affect the biogenesis and function of integral membrane proteins at multiple steps along its biogenetic pathway. While understanding this interdependence between bilayer lipids and protein biogenesis is interesting in its own right, careful consideration of a potential host’s membrane lipid composition may also allow optimization of the yield and activity of membrane proteins that are expressed in a heterologous organism. Here, we review and discuss some examples that illustrate the interdependence between bilayer lipids and the biogenesis of integral membrane proteins.     

Lipid Sorting and Multivesicular Endosome Biogenesis

Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions.It is now well established that membranes along the endocytic and secretory pathway show differences not only in protein but also in lipid composition. For example, lipid gradients exist along the biosynthetic pathway with increasing density of cholesterol and sphingolipids from the endoplasmic reticulum (ER) to the plasma membrane (Maxfield and van Meer 2010). Also, phosphoinositides show distributions restricted to relatively well-characterized membrane territories (Di Paolo and De Camilli 2006). Given the facts that lipids are small and contain little structural information when compared with proteins, that they can diffuse rapidly within membranes, and that membranes are connected by membrane flow during transport, it is not always obvious how different lipids are segregated from each other.In this article, we will evoke different mechanisms that may contribute to the heterogeneous lipid composition of endocytic membranes, including physicochemical properties of the membrane, interactions with other proteins or lipids, and synthesis or degradation. In addition, it has also become apparent that peripheral membrane proteins often interact with membranes via diverse lipid-binding motifs, and thus that lipids directly contribute to the distribution of many peripheral membrane proteins. For example, phosphatidylinositol 3-phosphate (PI(3)P) is detected predominantly on early endosomes, where most characterized PI(3)P-binding proteins encoded by the human genome are found as well (Raiborg et al. 2013). We will also discuss how some lipids may regulate protein sorting and membrane transport within the endosomal system.     

Lipid transport by mammalian ABC proteins

ABC (ATP-binding cassette) proteins actively transport a wide variety of substrates, including peptides, amino acids, sugars, metals, drugs, vitamins and lipids, across extracellular and intracellular membranes. Of the 49 hum an ABC proteins, a significant number are known to mediate the extrusion of lipids from membranes or the flipping of membrane lipids across the bilayer to generate and maintain membrane lipid asymmetry. Typical lipid substrates include phospholipids, sterols, sphingolipids, bile acids and related lipid conjugates. Members of the ABCA subfamily of ABC transporters and other ABC proteins such as ABCB4, ABCG1 and ABCG5/8 implicated in lipid transport play important roles in diverse biological processes such as cell signalling, membrane lipid asymmetry, removal of potentially toxic compounds and metabolites, and apoptosis. The importance of these ABC lipid transporters in cell physiology is evident from the finding that mutations in the genes encoding many of these proteins are responsible for severe inherited diseases. For example, mutations in ABCA1 cause Tangier disease associated with defective efflux of cholesterol and phosphatidylcholine from the plasma membrane to the lipid acceptor protein apoA1 (apolipoprotein AI), mutations in ABCA3 cause neonatal surfactant deficiency associated with a loss in secretion of the lipid pulmonary surfactants from lungs of newborns, mutations in ABCA4 cause Stargardt macular degeneration, a retinal degenerative disease linked to the reduced clearance of retinoid compounds from photoreceptor cells, mutations in ABCA12 cause harlequin and lamellar ichthyosis, skin diseases associated with defective lipid trafficking in keratinocytes, and mutations in ABCB4 and ABCG5/ABCG8 are responsible for progressive intrafamilial hepatic disease and sitosterolaemia associated with defective phospholipid and sterol transport respectively. This chapter highlights the involvement of various mammalian ABC transporters in lipid transport in the context of their role in cell signalling, cellular homoeostasis, apoptosis and inherited disorders.     

Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding

We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long-range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually dependent on each other for functional organization of proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Intramitochondrial phospholipid trafficking

Mitochondrial functions and architecture rely on a defined lipid composition of their outer and inner membranes, which are characterized by a high content of non-bilayer phospholipids such as cardiolipin (CL) and phosphatidylethanolamine (PE). Mitochondrial membrane lipids are synthesized in the endoplasmic reticulum (ER) or within mitochondria from ER-derived precursor lipids, are asymmetrically distributed within mitochondria and can relocate in response to cellular stress. Maintenance of lipid homeostasis thus requires multiple lipid transport processes to be orchestrated within mitochondria. Recent findings identified members of the Ups/PRELI family as specific lipid transfer proteins in mitochondria that shuttle phospholipids between mitochondrial membranes. They cooperate with membrane organizing proteins that preserve the spatial organization of mitochondrial membranes and the formation of membrane contact sites, unravelling an intimate crosstalk of membrane lipid transport and homeostasis with the structural organization of mitochondria.This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.     

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.