Felix T. Wieland

Molecular Anatomy of a Trafficking Organelle

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Ceramide Triggers Budding of Exosome Vesicles into Multivesicular Endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

Protein Sorting by Transport Vesicles

Eukaryotic life depends on the spatial and temporal organization of cellular membrane systems. Recent advances in understanding the machinery of vesicle transport have established general principles that underlie a broad variety of physiological processes, including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled release of hormones and neurotransmitters.

COUPLING OF COAT ASSEMBLY AND VESICLE BUDDING TO PACKAGING OF PUTATIVE CARGO RECEPTORS

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2FLAG, produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2FLAG particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2FLAG particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria.

Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

Molecular recognition of a single sphingolipid species by a protein's transmembrane domain.

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

The COPI system: Molecular mechanisms and function

Transport of membranes and proteins in eukaryotic cells is mediated by vesicular carriers. Here we review the biogenesis and functions of COPI vesicles, carriers that operate in the early secretory pathway. We focus on mechanisms mediating coat recruitment, uptake of cargo, vesicle budding and fission, and finally dissociation of the coat. In this context, recent findings on the interplay between machinery and auxiliary proteins in COPI vesicle formation and function will be discussed. Specifically, we will weigh the pros and cons of recent data on roles of the small GTP binding protein Arf1, of Arf1GAPs, and lipids during COPI carrier formation.

Sphingomyelin is synthesized in the cis Golgi

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi‐enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.

The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or “rafts” on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (≤3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ∼20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.