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F T Wieland

Publications and source records attributed to F T Wieland.

At least 19 recordsLinked to original sources
PubMed2001

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

ADP-Ribosylation Factor 1↗
PubMed2001

Sphingomyelin-enriched microdomains at the Golgi complex.

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Adenosine Triphosphatases↗
PubMed2000

Localization and topology of ratp28, a member of a novel family of putative steroid-binding proteins.

We have cloned ratp28, a membrane protein from rat liver homologous to the previously described hpr6.6, a putative steroid-binding protein in humans. Ratp28 has a type II topology as determined by protease digestion experiments on intact and detergent-solubilized membranes. Subcellular fractionation by sucrose density centrifugation revealed a distribution for ratp28 identical to Bip as a marker for membranes of the endoplasmic reticulum. In these experiments no association was found with markers for Golgi or plasma membranes, indicating that ratp28 is localized to the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed2000

Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry.

Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.

Amino Acid Sequence↗
PubMed2000

Structure of the cytoplasmic domain of p23 in solution: implications for the formation of COPI vesicles.

Coatomer, the coat protein complex of coat protein (COPI) vesicles, is involved in the budding of these vesicles. Its interaction with the cytoplasmic domains of some p24-family members, type I transmembrane proteins of the Golgi, has been shown to induce a conformational change of coatomer that initiates polymerization of the complex. From stoichiometrical data it is likely that interaction of coatomer with the small tail domains involves an oligomeric form of the p24 proteins. Here we present the structure of peptide analogs of the cytoplasmic domain of p23, a member of the p24 family, as determined by two-dimensional nuclear magnetic resonance spectroscopy in the presence of 2,2,2-trifluoroethanol. An improved strategy for structure calculation revealed that the tail domain peptides form alpha-helices and adopt a tetrameric state. Based on these results we propose an initial model for the binding of coatomer by p23 and the induced conformational change of coatomer that results in its polymerization, curvature of the Golgi membrane to form a bud, and finally a COPI-coated vesicle.

Amino Acid Sequence↗
PubMed2000

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles.

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.

ADP-Ribosylation Factor 1↗
PubMed1999

A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis.

ARF-mediated hydrolysis of GTP has been demonstrated to regulate coat disassembly of Golgi-derived COPI transport vesicles (Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) J. Cell Biol. 123, 1365-1371). In addition, a requirement for GTP hydrolysis at an early stage of COPI vesicle biogenesis has been established since cargo uptake is impaired in the presence of GTPgammaS (Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) J. Cell Sci. 111, 3081-3090), a non-hydrolyzable analogue of GTP. We now demonstrate that the GTPase involved in the regulation of cargo uptake is ARF, revealing a multi-functional role of this GTPase in COPI-mediated vesicular transport. The molecular mechanism of cargo uptake as well as the functional implications of these findings on the overall process of COPI vesicle biogenesis are discussed.

ADP-Ribosylation Factors↗
PubMed1999

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

Acetyltransferases↗
PubMed1999

GTP-dependent binding of ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23.

A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs.

ADP-Ribosylation Factors↗
PubMed1999

Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.

Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway.

COPI-coated vesicles that bud off the Golgi complex contain two major transmembrane proteins, p23 and p24. We have localized the protein at the Golgi complex and at COPI-coated vesicles. Transport from the intermediate compartment (IC) to the Golgi can be blocked at 15 degrees C, and under these conditions p24 accumulates in peripheral punctated structures identified as IC. Release from the temperature block leads to a redistribution of p24 to the Golgi, showing that p24, similar to p23, cycles between the IC and Golgi complex. Immunoprecipitations of p24 from cell lysates and from detergent-solubilized Golgi membranes and COPI-coated vesicles show that p24 and p23 interact with each other to form a complex. Transient transfection of p23 in HeLa cells shows that p23 and p24 colocalize in structures induced by the overexpression of p23. Taken together p24 interacts with p23 and constitutively cycles between the organelles of the early secretory pathway.

Amino Acid Sequence↗
PubMed1999

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.

ADP-Ribosylation Factors↗
PubMed1999

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.

Animals↗
PubMed1998

KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p.

A cholera toxin mutant (CTX-K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A-K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-gamma-S inhibits the internalization as well as Golgi-ER transport of CTX-A-K63. The appearance of CTX-A-K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, beta-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A-K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63-induced translocation of Erd2p and p53.

Adenosine Diphosphate Ribose↗
PubMed1998

A single binding site for dilysine retrieval motifs and p23 within the gamma subunit of coatomer.

Coatomer, the major component of the coat of COPI transport vesicles, binds both to the dilysine motif of resident membrane proteins of the endoplasmic reticulum and to the cytoplasmic domain of p23, a major type I membrane protein of COPI vesicles. Using a photocrosslinking approach, we find that under native conditions a peptide analogous to the cytoplasmic domain of p23 interacts with coatomer exclusively through its gamma subunit and shares its binding site with a KKXX retrieval motif. However, upon dissociation of coatomer, interaction with various subunits, including an alpha-, beta'-, epsilon-COP subcomplex, of the photoreactive peptide is observed. We suggest that, under physiological conditions, interaction of coatomer with both endoplasmic reticulum retrieval motifs and the cytoplasmic domain of p23 is mediated by gamma-COP.

Amino Acid Sequence↗
PubMed1998

A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles.

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.

Aluminum Compounds↗