scholarly journals Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro

Biology Open ◽  
2014 ◽  
Vol 3 (11) ◽  
pp. 1108-1115 ◽  
Author(s):  
N. Tamura ◽  
J. Mima
Keyword(s):  
Rab Gtpases ◽  
Membrane Tethering

scholarly journals Epstein-Barr Virus Exploits the Secretory Pathway to Release Virions

2020 ◽  
Vol 8 (5) ◽  
pp. 729
Author(s):  
Asuka Nanbo
Keyword(s):  
Epstein Barr Virus ◽  
Secretory Pathway ◽  
Lytic Cycle ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Extracellular Milieu ◽  
Barr Virus ◽  
Intracellular Compartments ◽  
Epstein Barr

Herpesvirus egress mechanisms are strongly associated with intracellular compartment remodeling processes. Previously, we and other groups have described that intracellular compartments derived from the Golgi apparatus are the maturation sites of Epstein-Barr virus (EBV) virions. However, the mechanism by which these virions are released from the host cell to the extracellular milieu is poorly understood. Here, I adapted two independent induction systems of the EBV lytic cycle in vitro, in the context of Rab GTPase silencing, to characterize the EBV release pathway. Immunofluorescence staining revealed that p350/220, the major EBV glycoprotein, partially co-localized with three Rab GTPases: Rab8a, Rab10, and Rab11a. Furthermore, the knockdown of these Rab GTPases promoted the intracellular accumulation of viral structural proteins by inhibiting its distribution to the plasma membrane. Finally, the knockdown of the Rab8a, Rab10, and Rab11a proteins suppressed the release of EBV infectious virions. Taken together, these findings support the hypothesis that mature EBV virions are released from infected cells to the extracellular milieu via the secretory pathway, as well as providing new insights into the EBV life cycle.

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals A role for Yip1p in COPII vesicle biogenesis

2003 ◽  
Vol 163 (1) ◽  
pp. 57-69 ◽  
Author(s):  
Matthew Heidtman ◽  
Catherine Z. Chen ◽  
Ruth N. Collins ◽  
Charles Barlowe
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Direct Role ◽  
Interacting Protein ◽  
Copii Vesicle ◽  
Vesicle Biogenesis

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

scholarly journals The structure of the Myo4p globular tail and its function in ASH1 mRNA localization

2010 ◽  
Vol 189 (3) ◽  
pp. 497-510 ◽  
Author(s):  
Alexander Heuck ◽  
Ingrid Fetka ◽  
Daniel N. Brewer ◽  
Daniela Hüls ◽  
Mary Munson ◽  
...  
Keyword(s):  
Mrna Localization ◽  
Surface Patch ◽  
X Ray ◽  
Membrane Tethering ◽  
Dividing Cells ◽  
Dependent Transport ◽  
Type V ◽  
Ash1 Mrna

Type V myosin (MyoV)–dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2–tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail–lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.

scholarly journals A role for Rab5 in structuring the endoplasmic reticulum

2007 ◽  
Vol 178 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Anjon Audhya ◽  
Arshad Desai ◽  
Karen Oegema
Keyword(s):  
Endoplasmic Reticulum ◽  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Rab Gtpases ◽  
C Elegans ◽  
Rab Family ◽  
Kinetics Of

The endoplasmic reticulum (ER) is a contiguous network of interconnected membrane sheets and tubules. The ER is differentiated into distinct domains, including the peripheral ER and nuclear envelope. Inhibition of two ER proteins, Rtn4a and DP1/NogoA, was previously shown to inhibit the formation of ER tubules in vitro. We show that the formation of ER tubules in vitro also requires a Rab family GTPase. Characterization of the 29 Caenorhabditis elegans Rab GTPases reveals that depletion of RAB-5 phenocopies the defects in peripheral ER structure that result from depletion of RET-1 and YOP-1, the C. elegans homologues of Rtn4a and DP1/NogoA. Perturbation of endocytosis by other means did not affect ER structure; the role of RAB-5 in ER morphology is thus independent of its well-studied requirement for endocytosis. RAB-5 and YOP-1/RET-1 also control the kinetics of nuclear envelope disassembly, which suggests an important role for the morphology of the peripheral ER in this process.

scholarly journals Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

2018 ◽  
Author(s):  
Lijun Guo ◽  
Marc Bramkamp
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Stress ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Organelle Division ◽  
Membrane Tethering ◽  
Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway

1999 ◽  
Vol 112 (24) ◽  
pp. 4773-4783 ◽  
Author(s):  
M. Trischler ◽  
W. Stoorvogel ◽  
O. Ullrich
Keyword(s):  
Intracellular Transport ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Recycling Endosomes ◽  
Morphological Differences ◽  
And Function ◽  
Cellular Compartments

Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.

scholarly journals Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210

2015 ◽  
Vol 26 (3) ◽  
pp. 537-553 ◽  
Author(s):  
Keisuke Sato ◽  
Peristera Roboti ◽  
Alexander A. Mironov ◽  
Martin Lowe
Keyword(s):  
Golgi Apparatus ◽  
Functional Significance ◽  
Coiled Coil ◽  
Rab Gtpases ◽  
Vesicle Tethering ◽  
Amino Terminal ◽  
Membrane Tethering ◽  
Per Se ◽  
Golgi Structure

Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210–mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus.

scholarly journals Electrical pulse stimulation induces GLUT4 translocation in C2C12 myotubes that depends on Rab8A, Rab13, and Rab14

2018 ◽  
Vol 314 (5) ◽  
pp. E478-E493 ◽  
Author(s):  
Zhu Li ◽  
Yingying Yue ◽  
Fang Hu ◽  
Chang Zhang ◽  
Xiaofang Ma ◽  
...  
Keyword(s):  
Muscle Contraction ◽  
Molecular Mechanisms ◽  
Electrical Pulse ◽  
C2c12 Myotubes ◽  
Rab Gtpases ◽  
Glut4 Translocation ◽  
Electrical Pulse Stimulation ◽  
Compound C ◽  
Pulse Stimulation

The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated C2C12 myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by small interfering (si)RNA-mediated AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca2+/calmodulin-dependent protein kinase (CaMKII)δ knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13, or Rab14 reduced, in part, GLUT4 translocation induced by EPS, whereas only Rab8a, or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13, and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

scholarly journals The Rod-Shaped ATG2A-WIPI4 Complex Tethers Membranes In Vitro

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641881993 ◽  
Author(s):  
Takanori Otomo ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Endoplasmic Reticulum ◽  
Critical Role ◽  
Critical Step ◽  
Molecular Events ◽  
Biochemical Studies ◽  
Membrane Tethering ◽  
Cytoplasmic Content ◽  
Autophagy Pathway ◽  
Copii Vesicles

The autophagosome precursor membrane, termed the isolation membrane or phagophore, emerges adjacent to a phosphatidylinositol 3-phosphate (PI3P)-enriched transient subdomain of the endoplasmic reticulum called the omegasome, thereafter expanding to engulf cytoplasmic content. Uncovering the molecular events that occur in the vicinity of the omegasome during phagophore biogenesis is imperative for understanding the mechanisms involved in this critical step of the autophagy pathway. We recently characterized the ATG2A-WIPI4 complex, one of the factors that localize to the omegasome and play a critical role in mediating phagophore expansion. Our structural and biochemical studies revealed that ATG2A is a rod-shaped protein with membrane-interacting properties at each end, endowing ATG2A with membrane-tethering capability. Association of the PI3P-binding protein WIPI4 at one of the ATG2A tips enables the ATG2A-WIPI4 complex to specifically tether PI3P-containing membranes to non-PI3P-containing membranes. We proposed models for the ATG2A-WIPI4 complex-mediated membrane associations between the omegasome and surrounding membranes, including the phagophore edge, the endoplasmic reticulum, ATG9 vesicles, and COPII vesicles.

Sign in / Sign up

Export Citation Format

Share Document