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‣ Proteínas da família FEZ (Fasciculation and Elongation protein Zeta) como adaptadoras bivalentes do transporte : aspectos funcionais, estruturais e evolutivos; FEZ proteins family (Fasciculation and Elongation protein Zeta) as bivalent transport adaptors : functional, structural and evolutionary aspects

Marcos Rodrigo Alborghetti
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 08/07/2011 Português
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As proteínas humanas FEZ1 e FEZ2 (fasciculation and elongation protein zeta) são ortólogas da proteína UNC-76 de C. elegans e estão envolvidas no crescimento e na fasciculação dos axônios através de interações que envolvem kinesinas, mitocôndrias e vesículas sinápticas. Além disso, algumas evidências sugerem a participação de FEZ1 na etiologia da esquizofrenia, no ciclo viral, além da resistência à quimioterápicos. Sua estrutura intrinsecamente desordenada, com coiled-coil ao longo da sequência, pode contribuir para sua função. Nós exploramos a evolução molecular da família de proteínas FEZ com ênfase no ramo dos vertebrados. Através do perfil do interactoma comparado entre FEZ1 e FEZ2 de Homo sapiens e UNC-76 de C. elegans foi observado um padrão de conservação das interações proteínaproteína entre FEZ1 e UNC-76, que explicam a capacidade de FEZ1 resgatar os defeitos causados por mutações em unc-76 em nematoides, de acordo com o descrito por Bloom e colaboradores em 1997. Além disso, caracterizamos a interação entre FEZ1 e SCOCO (short coil-coiled) por SAXS (Small Angle X-ray Scattering). Essa interação já foi descrita previamente entre os seus ortólogos UNC-76 e UNC-69, que cooperam no crescimento axonal. Um estado de heterotetramérico foi observado...

‣ Optical recording of signal-mediated protein transport through single nuclear pore complexes

Keminer, Oliver; Siebrasse, Jan-Peter; Zerf, Katja; Peters, Reiner
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 12/10/1999 Português
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Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, “patched” NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were ≈2 dimers/s for the import protein and ≈4 dimers/s for the export protein (≈15 μM substrate concentration, 22–24°C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.

‣ A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

Rieder, Stephanie E.; Emr, Scott D.
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /11/1997 Português
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Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPS gene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditional vps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition, vps18tsf cells accumulated multivesicular bodies...

‣ GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.

Baker, D; Wuestehube, L; Schekman, R; Botstein, D; Segev, N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1990 Português
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The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.

‣ Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

Hicke, L; Yoshihisa, T; Schekman, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1992 Português
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A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.

‣ Protein Binding by Specific Receptors on Human Placenta, Murine Placenta, and Suckling Murine Intestine in Relation to Protein Transport across These Tissues

Gitlin, Jonathan D.; Gitlin, David
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1974 Português
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Human, rat, and mouse placentas and rat and mouse intestines were homogenized in buffered saline, and fraction consisting primarily of cell membranes was separated from each of the homogenates by differential centrifugation. Human, bovine, and guinea pig IgG, and human IgE, Bence-Jones protein, serum albumin, insulin, and growth hormone were labeled with 131I or 125I, and the binding of these proteins by the cell membrane fractions was investigated. Rat and mouse sucklings were given labeled proteins intragastrically, and the amount of each protein absorbed after a given interval of time was determined. It was found that the degree and specificity of protein binding by the cell membrane fractions from human and murine placentas strikingly paralleled the relative rate and specificity of protein transport from mother to fetus in the respective species at or near term. Similarly, the degree and specificity of protein binding by the cell membrane fractions from suckling rat and mouse intestines tended to parallel the rate and specificity of protein absorption from the gastrointestinal tract in these animals. However, some discordance between protein binding and protein transport was also observed. The data suggest that: (a) the binding of a protein by specific receptors on cell membranes may be a necessary first step in the transcellular transport of the protein; (b) specific protein binding by cell receptors does not ensure the transport of that protein across the tissue barrier; and (c) specific transport mechanisms other than or in addition to specific cell membrane receptors are involved in the active transport of proteins across the human or murine placenta or the suckling murine intestine.

‣ A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Dierks, T; Volkmer, J; Schlenstedt, G; Jung, C; Sandholzer, U; Zachmann, K; Schlotterhose, P; Neifer, K; Schmidt, B; Zimmermann, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 16/12/1996 Português
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Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

‣ Effects of Picornavirus 3A Proteins on Protein Transport and GBF1-Dependent COP-I Recruitment▿

Wessels, Els; Duijsings, Daniël; Lanke, Kjerstin H. W.; van Dooren, Sander H. J.; Jackson, Catherine L.; Melchers, Willem J. G.; van Kuppeveld, Frank J. M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence...

‣ Temperature dependence of protein transport across lymphatic endothelium in vitro

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/02/1984 Português
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The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees...

‣ Basolateral protein transport in streptolysin O-permeabilized MDCK cells

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/06/1994 Português
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We have reconstituted polarized protein transport in streptolysin O- permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross- linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin- 2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together...

‣ Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/09/1995 Português
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Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein...

‣ Involvement of the Transmembrane Protein p23 in Biosynthetic Protein Transport

Rojo, Manuel; Pepperkok, Rainer; Emery, Gregory; Kellner, Roland; Stang, Espen; Parton, Robert G.; Gruenberg, Jean
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/12/1997 Português
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Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/μm2 membrane surface area, or ≈30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment . Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment...

‣ A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole

Darsow, Tamara; Rieder, Stephanie E.; Emr, Scott D.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 11/08/1997 Português
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Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift...

‣ A Stromal Pool of TatA Promotes Tat-dependent Protein Transport across the Thylakoid Membrane*

Frielingsdorf, Stefan; Jakob, Mario; Klösgen, Ralf Bernd
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 05/12/2008 Português
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In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.

‣ Immunocytochemical Evidence of Tulp1-dependent Outer Segment Protein Transport Pathways in Photoreceptor Cells

Grossman, Gregory H.; Watson, Rao F.; Pauer, Gayle J.T.; Bollinger, Kathryn; Hagstrom, Stephanie A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1−/− mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1−/− photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1−/− retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins...

‣ Protein Transport in Plant Cells: In and Out of the Golgi†

NEUMANN, ULLA; BRANDIZZI, FEDERICA; HAWES, CHRIS
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em /08/2003 Português
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In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glycosylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticulum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occurs via bulk flow. In this Botanical Briefing, we review the latest data on ER/Golgi inter‐relations and the models for transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER–Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass through the Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternae from the cis‐ to the trans‐face, a number of membrane proteins reside in the different Golgi cisternae. Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for the retention of proteins within the Golgi. In non‐dividing cells, protein transport out of the Golgi is either directed towards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lytic vacuole and protein storage vacuoles. In general...

‣ Characterisation of Tat protein transport complexes carrying inactivating mutations

McDevitt, C.; Hicks, M.; Palmer, T.; Berks, B.
Fonte: Academic Press Inc Publicador: Academic Press Inc
Tipo: Artigo de Revista Científica
Publicado em //2005 Português
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The Tat system functions to transport folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Tat transport involves a high molecular weight TatBC-containing complex that transiently associates with TatA during protein translocation. Sedimentation equilibrium experiments were used to determine a protein-only molecular mass for the TatBC complex of 630+/-30kDa, suggesting that it contains approximately 13 copies of the TatB and TatC protomers. Point mutations that inactivate Tat transport have previously been identified in each of TatA, TatB, and TatC. Analysis of the TatBC complexes formed by these inactive variants demonstrates that the amino acid substitutions neither affect the composition of the TatBC complex nor cause accumulation of the assembled TatABC translocation site. In addition, the TatA protein is shown not to be required for the assembly or stability of the TatBC complex.; Christopher A. McDevitt, Matthew G. Hicks, Tracy Palmer, Ben C. Berks

‣ The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter

Gohlke, U.; Pullan, L.; McDevitt, C.; Porcelli, I.; de Leeuw, E.; Palmer, T.; Saibil, H.; Berks, B.
Fonte: Natl Acad Sciences Publicador: Natl Acad Sciences
Tipo: Artigo de Revista Científica
Publicado em //2005 Português
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The Tat system mediates Sec-independent transport of folded precursor proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. Tat transport involves distinct high-molecular-weight TatA and TatBC complexes. Here we report the 3D architecture of the TatA complex from Escherichia coli obtained by single-particle electron microscopy and random conical tilt reconstruction. TatA forms ring-shaped structures of variable diameter in which the internal channels are large enough to accommodate known Tat substrate proteins. This morphology strongly supports the proposal that TatA forms the protein-conducting channel of the Tat system. One end of the channel is closed by a lid that might gate access to the channel. On the basis of previous protease accessibility measurements, the lid is likely to be located at the cytoplasmic side of the membrane. The observed variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of the channel to the size of the substrate being transported. Such dynamic close packing would provide a mechanism to maintain the membrane permeability barrier during transport.; Ulrich Gohlke, Lee Pullan, Christopher A. McDevitt, Ida Porcelli...

‣ Potenzierung der Suizidgen-Wirkung durch VP22-Fusionsproteine; Enhanced suicide gene effect with VP22 fusion proteins

Wybranietz, Wolfgang
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Die Suizidgen-Therapie versucht Tumorzellen, in die Selbstmord-gene wie Cytosin-Desaminase (CD) aus E. coli oder Thymidin-Kinase (TK) aus Herpes simplex-Virus (HSV-1) eingebracht wurden, spezifisch abzutöten, ohne das umgebende Gewebe zu schädigen. In vivo ist die Gentransfer-Effizienz jedoch zu gering, um wirksame Mengen der Selbstmord-Proteine zu bilden, so dass Tumore bislang nicht eliminiert werden konnten. Das HSV-1 Protein VP22 kann Proteine aus einer Produzenten-zelle in bis zu 500 Nachbarzellen transportieren, so dass die Zahl der erreichten Zellen durch VP22-Transport (spread) entscheidend vergrößert wird. Durch Quantifizierung des spread eines VP22-GFP-Fusionsproteins in 15 Säugerzelllinien mittels FACS wurde zunächst gezeigt, dass es sich beim VP22-vermittelten inter-zellulären Proteintransport um ein generelles biologisches Phänomen handelt. Darauf aufbauend wurde die Wirkungssteigerung in N- und C-ter-mi-na-len Fusionen der Suizidgene CD und TK mit VP22 in adenoviralen Vektoren überprüft. Nach Transfektion von Expressionsplasmiden ergab sich, dass TK-Fusionskonstrukte mit VP22 in beiden Verknüpfungen exzellenten Proteintransport aufwiesen, während CD sich nur als CD-VP22-Fusion transportieren ließ. Mittels Immunfluoreszenzfärbung wurde gezeigt...

‣ Mechanisms of vacuolar protein transport in plants

Künzl, Fabian
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
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Protein transport to plant vacuoles can occur by the biosynthetic pathway, leading from the endoplasmic reticulum (ER) via the Golgi apparatus, or by the endocytic pathway from the plasma membrane (PM). Both routes converge in the trans-Golgi network/early endosome (TGN/EE), from where cargo passes a multivesicular late endosome (MVB/LE) before reaching the vacuole. PM proteins destined for vacuolar degradation are internalized into the intraluminal vesicles of MVBs/LEs in a process involving recognition and sorting by the ESCRT machinery. Soluble vacuolar proteins from the biosynthetic pathway have to interact with vacuolar sorting receptors (VSRs) to be diverted from the secretory pathway and sent into the vacuolar route. Regarding these transport events, several aspects have remained unclear: how are proteins transported from the TGN/EE to the MVB/LE, and how do they arrive in the vacuole? Where does ESCRT recognize its cargo, and when does it initiate intraluminal sorting? What is the sorting signal for entry into the ESCRT-mediated vacuolar pathway? Where in the endomembrane system do VSRs bind soluble cargo ligands and where do they release them? In this thesis, results from two publications and a currently submitted manuscript are presented. We established that “multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis” (Scheuring et al....