Membrane Localization

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Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence

Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence

Cardiolipin synthesis is required for plaque formation. To determine the role of cardiolipin in S. flexneri pathogenesis, we performed plaque assays (47) with each of the cardiolipin synthesis mutants. Plaque formation required invasion of the monolayer, intracellular replication, and spread to the adjacent cells. After 72 h, the clsA mutant formed pinpoint plaques compared to the WT, and plaque formation was comple- mented by clsA on a plasmid (Fig. 3). Because ClsC contributed to cardiolipin synthesis in stationary phase, it was possible that the ClsB or ClsC was expressed in the intracellular environment and contributed sufficient cardiolipin to support formation of very small plaques. Both clsB and clsC were induced approximately 10-fold in intracel- FIG 2 S. flexneri requires pbgA for localization of cardiolipin to its outer membrane. TLC analysis of S. flexneri inner and outer membrane phospholipids. (A) Inner and outer membrane composition analysis of pbgA and clsA mutants. Bacteria were grown to mid-log phase, and bacterial membranes were separated by solubilization in Sarkosyl. The phospholipids were then extracted, spotted for separation by TLC, and visualized using molybdenum blue spray reagent (Sigma). The phospholipids include phos- phatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL). The percentage of anionic phospholipids (AL) in the sample is indicated above each lane, and means ⫾ standard deviations of three experiments are shown. The value that is significantly different (P ⬍ 0.05) from the value for the outer membrane of the wild type by Student’s t test is indicated by an asterisk. (B) Confirmation of clean inner and outer membrane fractions. Bacteria were grown to mid-log phase, and the bacterial membranes were separated by solubilization using Sarkosyl. Proteins were resolved by (10%) SDS-PAGE and stained with Coomassie blue (CB) or immunoblotted using either polyclonal anti-SecA or polyclonal anti-OmpA. The molecular masses (in kilodaltons) of molecular mass markers are shown to the left of the gels. ppbgA, plasmid expressing pbgA; C, cytoplasm; IM, inner membrane; OM, outer membrane.
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Plasma membrane localization of MLC1 regulates cellular morphology and motility

Plasma membrane localization of MLC1 regulates cellular morphology and motility

we examined whether the subcellular localization of wildtype MLC1 can affect alterations in both morph- ology and motility during the surface-targeting process. Indeed, some cells transfected with wildtype MLC1 also exhibited mutant-like intracellular localization of MLC1 proteins. From the randomly selected regions of interest (ROIs), 17.5% ( n = 30) of cells showed that the wildtype MLC1 proteins was trapped intracellularly with a small fraction of surface expression at 24 h after transfection. We classified MLC-expressing cells based on the subcellu- lar distribution of MLC1 proteins: PM-MLC1 cells, in which MLC1 is found at the plasma membrane, and ER- MLC1 cells, in which the significant fraction of MLC1 proteins are trapped in the ER. To detect surface- expressed MLC1 accurately, we placed the Myc-tag in the putative extracellular loop between the third and the fourth transmembrane domains. Immunofluorescence staining confirmed the exclusive accessibility of the Myc- tag from the extracellular side (Additional file 3: Figure S3). Interestingly, the PM-MLC1 cells exhibited abnormal spiky membrane protrusions with the significant surface expression of MLC1 (Fig. 4a, marked with triple asterisks), while the ER-MLC1 cells exhibited normal morphology with feeble MLC1 signals at the cell surface (Fig. 4a, an as- terisk), which is somewhat similar to cells expressing patient-derived mutants (Fig. 2a). An intermediate distri- bution of MLC1 with weak surface MLC1 signals and relatively few filopodia was also observed (Fig. 4a, double asterisks). Moreover, the polarized distribution of surface MLC1 resulted in asymmetric filopodia formation: The re- gion with higher surface expression of MLC1 induced well-developed filopodia formation while reduced surface expression could not. These asymmetric filopodia forma- tion indicates that localization of MLC1 at the plasma membrane is critical to the morphological changes of the cell membrane (Fig. 4b). We also compared cellular motil- ity between PM- and ER-MLC1 cells via live-cell imaging over 500 min. Kymographs of the PM- and ER-MLC1 cells indicated that cellular motility is regulated by the subcel- lular localization of MLC1 (Fig. 4c). The mean velocity of ER-MLC1 cells was significantly higher than that of PM- MLC1 cells (Fig. 4d), although no changes in directionality were observed as in the cases of patient-derived MLC1 mutants (Fig. 4e). The live-cell imaging allowed us to gain
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Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

LMP has been shown to function in these cell lines 2, 10; in HEp-2 cells LMP is cytotoxic, and in BALB/c 3T3 cells LMP induces both anchorage-independent growth at moderate levels of exp[r]

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Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In mammalian cells, multiple Ras isoforms have been char- acterized, which include H-Ras, N-Ras, and K-RasB. These Ras isoforms share almost identical amino acid sequences but are functionally distinct (21). Recent studies have found that functional specificity of these isoforms is dictated at least in part by differential membrane localization, which in turn is due to posttranslational modifications within a C-terminal hyper- variable region (HVR) that culminates with the CAAX motif (18, 21). Initial membrane association occurs by the prenyla- tion of a specific cysteine residue in the CAAX motif, targeting Ras to the endoplasmic reticulum (ER). Inhibition of Ras prenylation results in defective membrane association and im- paired protein function. Consistent with the observation that prenylation is required for Ras activity, this process is consti- tutive and irreversible (35).
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Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40

While some structure-function data on eVP40 are available, almost nothing is known about mVP40 membrane interactions. Comparison of the eVP40 and mVP40 sequences revealed amino acid sequence identity of 34%, with even less identity in the CTD membrane binding region. These sequence dissimilarities seem to contribute to the different lipid binding properties of eVP40 and mVP40. Previous studies have demonstrated that eVP40 can as- sociate with vesicles containing PS (20, 22–24, 38). While these previous studies have not discriminated between specific and nonspecific electrostatic interactions with PS, more detailed ex- periments with PS interactions in cell culture have demonstrated that PS plays an important and specific role in interacting with eVP40 (59, 60). Additionally, treatment of human and mamma- lian cell lines expressing eVP40 with sphingosine did not have a significant effect on eVP40 plasma membrane localization (59, 60). These studies have strongly suggested that eVP40 membrane association is not a simple case of anionic charge sensing. This suggests that though mVP40 and eVP40 both rely heavily on elec- trostatic interactions for membrane association, there is a funda- mental difference between the two proteins’ mechanism of assem- bly at the plasma membrane. Many peripheral proteins rely on
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Na/K/Cl co transport and its regulation

Na/K/Cl co transport and its regulation

In these tissues, luminal but not serosal exposure to 'loop' diuretics results in an inhibition of Na and Cl influx into the epithelium, confirming an apical membrane localization for Na[r]

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Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

In this paper, we describe the plasma membrane localization of P68 in UR2-transformed cells by subcellular fractionation and characterize differences between its membrane association and[r]

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Integrins (alpha7beta1) in muscle function and survival  Disrupted expression in merosin deficient congenital muscular dystrophy

Integrins (alpha7beta1) in muscle function and survival Disrupted expression in merosin deficient congenital muscular dystrophy

Mutations in genes coding for dystrophin, for a , b , g , and d -sarcoglycans, or for the a 2 chain of the basement mem- brane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an ab- normal expression and localization of a 7 b 1 isoforms in myo- fibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483–1497). Correction of merosin defi- ciency in vitro through cell transfection with the merosin a 2 chain restored the normal localization of a 7 b 1D integrins as well as myotube survival. Overexpression of the apopto- sis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of a 7 b 1D integrins. Blocking of b 1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings ( a) identify a 7 b 1D integrins as the de facto receptors for merosin in skeletal muscle; ( b) indicate a merosin dependence for the accurate expression and membrane localization of a 7 b 1D integrins in myofi- bers; ( c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. ( J. Clin. In- vest. 1997. 100:1870–1881.) Key words: apoptosis • base-
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The deubiquitinating enzyme USP17 is essential for GTPase subcellular localization and cell motility

The deubiquitinating enzyme USP17 is essential for GTPase subcellular localization and cell motility

Therefore, we assessed the effect of USP17 depletion on the intracel- lular localization and activation of Rac1, RhoA and Cdc42 in the context of cell migration. In unstimulated cells, these GTPases are localized primarily to the cytosolic regions of the cells. Following CXCL12 stimulation in control scrambled shRNA cells, the GTPases are transported to the plasma membrane (Fig. 5a–c white arrows). However, in USP17-depleted cells (shRNA1), the plasma membrane localization of Cdc42, Rac1 and RhoA was markedly blunted (Fig. 5a–c, respectively). In addition, USP17 shRNA2 also resulted in a mislocalization of RhoA, whereas CYLD shRNA had no effect on its transport (Supplementary Fig. S5). The localization of endogenous RhoA, Cdc42 and Rac1 was also examined and a similar trend was observed such that USP17 was required for GTPase membrane local- ization following chemokine stimulation (Supplementary Fig. S6). Fluorescent images were overlayed on brightfield images to confirm
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Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

prepared by sequential extraction with boiling SDS-containing buffer followed by treatment with Quantazyme, a fourth, still higher-molecular-mass species (⬎300 kDa) of Aga1p was re- leased. It is predominantly, if not only, this form of Aga1p that cross-reacts with antibodies specific to ␤-1, 6 glucan (Fig. 3B). Thus, our data indicate that at least four forms of Aga1p are FIG. 3. Aga1p is a GPI-anchored plasma membrane and cell wall-linked protein. (A) Immunoblot detection of Aga1p in total cell lysates and material releasable from intact cells by Quantazyme or SDS extraction. Total cell lysates of aga1⌬ cells carrying either an empty vector (pRS315) or the 3HA-tagged Aga1p-encoding plasmid (pAGA1) were obtained and immunoblotted as described in Materials and Methods. Quantazyme and SDS treatment of intact aga1⌬ cells carrying pAGA1 released the highest- and middle-molecular-mass species of Aga1p (marked by ⬎). (B) Immunoblot of Quantazyme-releasable Aga1p from purified cell wall materials. The Quantazyme-releasable proteins were immunoprecipi- tated with HA agarose beads and detected with either anti-HA or anti-␤-1,6 glucan antibodies. (C) Immunoblot of total cell lysates derived from strains expressing the chimeric Aga1 proteins shown in Fig. 1A and 2A. Twenty-microliter samples were loaded for lanes 1, 2, 3, and 8, and 2-␮l samples were loaded for lanes 4 to 7. A separate blot of the same lysates (2 ␮l) was probed with anti-PSTAIR antibodies and detected for the levels of Cdc28p as a control for equivalence of total protein in lysates. (D) A pool of Aga1 proteins cross-reacts with antibody specific to the cell wall ␤-1,6 glucan in all of the GPI-anchored Aga1p chimeras. Samples were treated similarly, as indicated in panel B. The upper panel shows a representative image of the immunoblot analysis. Lanes are loaded with equivalent amounts of immunoprecipitated material, except lane 3, the loading of which is ⬃0.8⫻ that of the other samples due to a correspondingly reduced starting amount of cells used for extraction. The lower panel shows the amount of Quantazyme-releasable Aga1p in the chimeras relative to the wild-type (WT) Aga1p. The data shown are the average from two independent experiments; levels of the Aga1p::Gas1-GPI are normalized to the other samples. Histogram columns 1 to 4 represent pAGA1, pAGA1-(GPI-CWP2), pAGA1-(GPI-GAS1), and pAGA1-(GPI-YAP3), respectively.
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p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

What is the significance of p27 localization to rafts within the plasma membrane? Because p27 is excluded when rafts aggregate into large signaling complexes, it is conceivable that p27 antagonized signaling. p27 could be a component of a complex that helps maintain lymphocytes in a basal state of quiescence. This could occur through inhibition of kinases, a function suggested for membrane- associated p27 in rat liver (50). Alternatively, the presence of p27 could forestall aggregation of raft complexes in quiescent cells. In a natural killer (NK) cell model, inhibition of raft aggregation by tumor cell major histocompatibility complex (MHC) recep- tors attenuated NK cell function (24). Future inves- tigations should uncover whether p27 modulates raft-based signal transduction and, if so, whether this differs between normal and neoplastic cells. antibody. Changes in the makeup of detergent-
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Gomes Rocha, Agostinho Manuel
  

(2012):


	Calcium regulation in chloroplasts and the role of calcium-dependent phosphorylation of transketolase in carbon metabolism.


Dissertation, LMU München: Fakultät für Biologie

Gomes Rocha, Agostinho Manuel (2012): Calcium regulation in chloroplasts and the role of calcium-dependent phosphorylation of transketolase in carbon metabolism. Dissertation, LMU München: Fakultät für Biologie

In order to identify candidates, “cold” phosphorylation assays were performed with the same samples. Protein spots were excised out of the coomassie brilliant blue stained gel from the regions of interest and proteins were analyzed by mass spectrometry. As was to be expected from such a complex mixture of proteins, peptides were found that matched several different proteins, a list of which can be found as Annex Table 2. The candidate list was filtered based on the estimated molecular mass (excluding cleaved targeting peptides), the theoretical isoelectric point of the mature protein (Wilkins et al. 1999) and predicted chloroplastidic localization according to the Aramemnon database (Schwacke et al. 2003). Moreover, the list only displays proteins for which a phosphopeptide was previously identified in phosphoproteomic approaches (PhosPhAt database, Durek et al. 2010; Heazlewood et al. 2008).
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Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4.

Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4.

Specific localization of the lysis protein of bacteriophage MS2 in membrane adhesion sites of Escherichia coli.. In vivo synthesis and properties of uracil-containing DNA.[r]

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Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein

Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein

drial functions that require the expression of additional SARS- CoV-encoded proteins, as would occur in a natural infection. An important role for ORF 3b as an inhibitor of type I IFN expression has recently been described (25). In a previous study, overexpression of ORF 3b was shown to inhibit IRF-3 phosphorylation, reduce expression from an IFN-stimulated response element-driven reporter plasmid, and allow for rep- lication of a type I IFN-sensitive virus, Newcastle disease virus. The mechanism by which ORF 3b blocks type I IFN produc- tion is unknown. MAVS is targeted by several virally encoded interferon antagonists (6, 28, 38). It is possible that ORF 3b targets this signaling pathway when it has reached the outer membrane of mitochondria. Work by Spiegel and colleagues demonstrated that during SARS-CoV infection, IRF-3 initially translocates to the nucleus at 8 h postinfection but is found redistributed in the cytoplasm at 16 h postinfection and fails to induce IFN- ␤ expression (34). It is interesting that ORF 3b may influence this process since dynamics of ORF 3b nucleo- mitochondrial translocation occur within a similar time frame. Thus, our elucidation of the spatiotemporal distribution of the SARS-CoV ORF 3b protein may be important in understand- ing the mechanism by which SARS-CoV inhibits the IFN re- sponse.
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Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

Oscillation of STEVOR expression and differential localization. Biphasic gene expression of STEVORs could enable a single variant to perform different functions during the IDC. We confirmed biphasic gene expression on the protein level using specific antisera for four selected STEVORs with similar gene expression profiles: PF3D7_0631900, PF3D7_1254100, PF3D7_0324600, and PF3D7_0300400 (Fig. 2B). Se- quence specificity of the antisera was confirmed using transgenic parasite lines ex- pressing the selected STEVOR as either green fluorescent protein (GFP)- or hemagglu- tinin (HA)-tagged variants (Fig. S5A to C). For semiquantitative Western blot analysis, parasite lysates were harvested in parallel to RNA samples, and signal intensities were quantified densitometrically and normalized to a glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) loading control (Fig. 2C). The antisera detected a single protein of approximately 35 kDa in Western blot analysis corresponding to the calculated molec- ular weight of 35 to 37 kDa (Fig. 2B). These results mirror the RNA expression pattern but with a time delay in protein abundance caused by translational processes and show a biphasic protein expression pattern with the highest protein levels in early tropho- zoites (24 h postinfection), a subsequent drop in protein levels, and then a gradual increase of the signal intensity until the late schizont stage. A significant amount of STEVOR protein was also found in purified merozoites but to a lesser extent than that in schizonts.
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Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

FIG 1 Eyespot location and structure. (A) Diagram of a Chlamydomonas cell highlighting the eyespot (red oval) adjacent to the daughter four-membered microtubule rootlet (D4). The D4 is one of four bundles of acetylated microtubules that extend from the two mature basal bodies at the anterior end of the cell (blue circles). The two basal bodies differ in age; the daughter basal body, in the cis position relative to the eyespot, matured just prior to the most recent cell division, while the older mother basal body, in the trans position relative to the eyespot, matured prior to an earlier division. Each basal body is associated with one rootlet that comprises four microtubules (D4 and M4, thick blue lines) and one rootlet that comprises two microtubules (D2 and M2, thin blue lines). The rootlets lie just under the plasma membrane and are approximately 90° from one another, forming a cruciate structure; the dashed lines represent the two rootlets that extend away from the viewer. (B) Left, diagram of postmitotic daughter cells in which new D4 rootlets (solid light-blue lines) have extended from the new daughter basal bodies (light-blue circles outlined with dark blue) in a plane that is perpendicular to the cleavage plane, and the eyespots have assembled de novo adjacent to the D4 rootlets. Right, eyespots comprise layers of carotenoid pigment granules (red circles) subtended by thylakoid membranes (TM) in the chloroplast, closely apposed to the chloroplast envelopes (CE) and the plasma membrane (PM). Channelrhodopsin photoreceptors ChR1 and ChR2 (dark-red ovals) span the plasma membrane, and EYE2 (small blue rectangles) is associated with one of the two chloroplast envelopes. (C) Summary of Chlamydomonas cell division focusing on microtubules. Interpretive line drawings are above representative indirect immunofluorescence (IF) images of cells viewed from the anterior pole: green, ␣ -Ac-tub; red, ␣ -tubulin (ii to iv) or anti-ChR1 (i and v). (i) Interphase cells have
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Spatial regulation of exocytosis by Rho family small GTPases in Saccharomyces cerevisiae

Spatial regulation of exocytosis by Rho family small GTPases in Saccharomyces cerevisiae

In contrast to the consistently dispersed pattern of localization found for Rho3, Cdc42 is tightly polarized to bud tips during bud emergence but this polarized staining is lost as the bud enlarges. This pattern of localization appears to require a distinct set of signals from that of Rho3 that are likely to reside in the C-terminal portion of the protein. In addition to C- terminal prenylation (CAAX) signal a stretch of five basic residues, known as a poly basic region is present at the C-terminus of Cdc42. The pattern of localization observed for Cdc42 closely mirrors the pattern observed for many proteins which ride to the plasma membrane along with polarized delivery of post-Golgi secretory vesicles. This list includes Sec4, Sec2, Myo2, and most of the subunits of the exocyst complex. Consistent with this notion Cdc42 has been suggested to itself be associated with post-Golgi secretory vesicles during cell fractionation (Wedlich-Soldner et al., 2003) and its polarized localization is rapidly lost with a block in polarized secretion (Irazoqui et al., 2003) (Zhang et al., 2001) The switch of function chimera, Cdc42-NT R3 , appears to have the ability to adopt both Rho3-like and Cdc42-like patterns of localization. This suggests that the pathways that mediate these two localization patterns operate independently of each other. The effects of the palmitoylation site mutation on the localization of Cdc42-NT R3 support this view—as we see the loss of
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Rab6 mediates membrane organization and determinant localization during
Drosophila oogenesis

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

rab6 D23D egg chambers that develop past stage 7 display phenotypes ranging from wild type to a loss of nurse cell cortical actin and the concomitant presence of ring canal clusters in the nurse cell cytoplasm (Murthy and Schwarz, 2004) (this study). The striking parallel between the rab6 and sec5 phenotypes, together with our finding that a loss of Rab6 affects Sec5 localization, suggests that the varying degrees of membrane loss observed in rab6 D23D egg chambers reflects the relative reduction of exocyst-complex function in the egg chamber. Thus, during Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore appears to be important for exocyst-complex organization and function. However, consequent to loss of rab6 function, we observed a striking difference between nurse cells and oocyte in the severity of plasma membrane collapse and Sec5 mislocalization. We hypothesize that the oocyte acts as a major source of membrane in rab6 D23D egg chambers and/or that multiple exocytic pathways cooperate within the germline cyst to promote cyst development.
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Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents

Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents

When any of these reagents was reacted with virus in the presence of 1 mg of cytochrome c per ml, there was no labeling of the cytochrome c data not shown, indicating that this system of[r]

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Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and Localization of Virion Membrane Proteins to Sites of Virion Assembly

Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and Localization of Virion Membrane Proteins to Sites of Virion Assembly

The A6L inducible mutant also showed a novel phenotype that has not been previously reported for VACV mutants that are de- fective in virion membrane biogenesis. During normal VACV in- fection, MV membrane proteins predominantly localize to viral factories, where virion assembly occurs. The intracellular localiza- tion of MV membrane proteins in the absence of A11, H7, or L2 was previously studied by immunofluorescence analysis of MV membrane protein L1 or A17, and no defect was observed (21, 38). In contrast, when A6L expression was repressed, there was a pro- found defect in localization of MV membrane proteins to viral factories. This defect was demonstrated with four different MV membrane proteins that were inserted into membranes through two different mechanisms. A13 and A14 were presumably synthe- sized on rough ER and cotranslationally inserted into membranes (37), while H3 and presumably D8 were synthesized on free ribo- somes and posttranslationally inserted into membranes (4). The absence of A6 did not affect the levels of these four MV proteins in the cells but dramatically affected their intracellular localization. Immunofluorescence analysis showed that they no longer local- ized to viral factories but instead colocalized extensively with ER markers PDI and calnexin outside the factories (Fig. 4, 5, and 6). A13 also accumulated in areas that were costained for ERGIC53, a marker for ERGIC. Furthermore, cell fractionation studies showed that, in the absence of A6, only a small amount of these MV proteins sedimented with virions, while the majority of the proteins sedimented at densities that were lighter than virions and were similar to that of the ER (Fig. 7).
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