Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis.

Endosomes play a major role in various cellular processes including cell-cell signaling, development and cellular responses to environment. Endosomes are dynamically organized into a complex set of endomembrane compartments themselves subcompartmentalized in distinct pools or subpopulations. It is increasingly evident that endosome dynamics and maturation is driven by local modification of lipid composition. The diversity of membrane lipids is impressive and their homeostasis often involves crosstalk between distinct lipid classes. Hence, biochemical characterization of endosomal membrane lipidome would clarify the maturation steps of endocytic routes. Immunopurification of intact endomembrane compartments has been employed in recent years to isolate early and late endosomal compartments and can even be used to separate subpopulations of early endosomes. In this section, we will describe the immunoprecipitation protocol to isolate endosomes with the aim to analyze the lipid content. We will detail a procedure to identify the total fatty acid and sterol content of isolated endosomes as a first line of lipid identification. Advantages and limitations of the method will be discussed as well as potential pitfalls and critical steps.

Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor.

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.

Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin.

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

Glycosylphosphatidylinositol-anchored proteins: Membrane organization and transport.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of membrane proteins containing a soluble protein attached by a conserved glycolipid anchor to the external leaflet of the plasma membrane. In polarized epithelial cells, GPI-APs are predominantly sorted to the apical surface in the trans-Golgi network (TGN) by clustering in sphingolipid- and cholesterol-dependent microdomains (or rafts), which have been proposed to act as apical sorting platforms. Recent data indicate that the mechanisms of GPI-AP sorting, occurring in the Golgi, control both the membrane transport of GPI-APs and their specific activity at the apical surface of fully polarized epithelial cells. Here, we discuss the most recent findings and the factors regulating apical sorting of GPI-APs at the Golgi in polarized epithelial cells. We also underline the differences in the plasma membrane organization of GPI-APs between polarized and non-polarized cells supporting the existence of various mechanisms that control GPI-AP organization in different cell types.

Itinerary of high density lipoproteins in endothelial cells.

High density lipoprotein (HDL) and its main protein component apolipoprotein A-I (ApoA-I) have multiple anti-atherogenic functions. Some of them are exerted within the vessel wall, so that HDL needs to pass the endothelial barrier. To elucidate their itinerary through endothelial cells (ECs), we labelled ApoA-I and HDL either fluorescently or with 1.4 nm nanogold and investigated their cellular localization by using immunofluorescent microscopy (IFM) and electron microscopy (EM). HDL as well as ApoA-I is taken up by ECs into the same route of intracellular trafficking. Time kinetics and pulse chase experiments revealed that HDL is trafficked through different vesicles. HDL partially co-localized with LDL, albumin, and transferrin. HDL did not co-localize with clathrin and caveolin-1. Fluorescent HDL was recovered at small proportions in early endosomes and endosome to trans-golgi network vesicles but not at all in recycling endosomes, in late endosomes or lysosomes. EM identified HDL mainly in large filled vesicles which however upon IFM did not colocalize with markers of multivesicular bodies or autophagosomes. The uptake or cellular distribution of HDL was altered upon pharmacological interference with cytochalasine D, colchicine and dynasore. Blockage of fluid phase uptake with Amiloride or EIPA did not reduce the uptake of HDL. Neither did we observe any co-localization of HDL with dextran as the marker of fluid phase uptake. In conclusion, HDL and ApoA-I are internalized and trafficked by endothelial cells through a non-classical endocytic route.

Modeling Yeast Organelle Membranes and How Lipid Diversity Influences Bilayer Properties.

Membrane lipids are important for the health and proper function of cell membranes. We have improved computational membrane models for specific organelles in yeast Saccharomyces cerevisiae to study the effect of lipid diversity on membrane structure and dynamics. Previous molecular dynamics simulations were performed by Jo et al. [(2009) Biophys J. 97, 50-58] on yeast membrane models having six lipid types with compositions averaged between the endoplasmic reticulum (ER) and the plasma membrane (PM). We incorporated ergosterol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol lipids in our models to better describe the unique composition of the PM, ER, and trans-Golgi network (TGN) bilayers of yeast. Our results describe membrane structure based on order parameters (SCD), electron density profiles (EDPs), and lipid packing. The average surface area per lipid decreased from 63.8 ± 0.4 Å(2) in the ER to 47.1 ± 0.3 Å(2) in the PM, while the compressibility modulus (KA) varied in the opposite direction. The high SCD values for the PM lipids indicated a more ordered bilayer core, while the corresponding lipids in the ER and TGN models had lower parameters by a factor of at least 0.7. The hydrophobic core thickness (2DC) as estimated from EDPs is the thickest for PM, which is in agreement with estimates of hydrophobic regions of transmembrane proteins from the Orientation of Proteins in Membranes database. Our results show the importance of lipid diversity and composition on a bilayer's structural and mechanical properties, which in turn influences interactions with the proteins and membrane-bound molecules.

Topological regulation of lipid balance in cells.

Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient.

Cellular localization and biochemical characterization of a chimeric fluorescent protein fusion of Arabidopsis cellulose synthase-like A2 inserted into Golgi membrane.

Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of ß -mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-D-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.

Interaction of cisplatin and analogue Pt(en)Cl2 with the copper metallo-chaperone Atox1.

The human metallo-chaperone protein Atox1 features a high affinity Cu(I) binding site Cys(12)GlyGlyCys(15) (KD = 10(-17.4) M at pH 7.0) and delivers copper to the trans-Golgi network (TGN). Atox1 may participate in the metabolism of the drug cis-Pt(NH3)2Cl2 (cisplatin), either as a component of its delivery to the nucleus or of its loss via transport to the TGN and beyond. The species of stoichiometry [Pt(NH3)2(Atox1)] was the sole adduct of stoichiometry Pt : Atox1 = 1 : 1 detected by mass spectrometry under non-denaturing conditions from solutions containing cisplatin and apo-Atox1. The ions [Atox1 + Pt(NH3)2(2+) + (z - 2)H(+)](z+) (z = 3 to 7) were observed and correspond to different protonation states of the 1 : 1 adduct. Adducts of stoichiometry Pt : Atox1 = 2 : 1 were also detected but 1 : 2 adducts were not detected. The related complex Pt(en)Cl2 (en = 1,2-diaminoethane) behaved similarly. Tandem mass spectrometry experiments using top-down and bottom-up sequencing techniques were carried out, respectively, on the intact platinated protein and on platinated peptides formed from proteolysis by trypsin. A new software programme (PolyCut) designed to analyse the complex high-resolution tandem mass spectra of fragment ions derived from proteins containing transition metal ions was applied to establish the binding site(s) of the platinum atom(s). The analysis, based on the entire isotope patterns, is consistent with the cysteine residues in the Cu(I)-binding sequence Cys(12)GlyGlyCys(15) being the primary coordination site.

The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 µM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.