scholarly journals Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane

2014 ◽  
Vol 7 (12) ◽  
pp. 1335-1340 ◽  
Author(s):  
H. Brandstaetter ◽  
A. J. Kruppa ◽  
F. Buss
Keyword(s):  
Plasma Membrane ◽  
Secretory Vesicle ◽  
Vesicle Fusion ◽  
Golgi Transport

scholarly journals Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation

2021 ◽  
Author(s):  
Martin Alejandro Pavarotti ◽  
Victoria Tokarz ◽  
Scott Frendo-Cumbo ◽  
Philip Bilan ◽  
Zhi Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Signalling ◽  
Muscle Cells ◽  
Secretory Vesicle ◽  
Vesicle Fusion ◽  
Glut4 Translocation ◽  
Vesicle Membrane ◽  
Actin Fiber ◽  
Complexin 2 ◽  
Intracellular Compartments

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane. GLUT4 vesicle fusion requires formation of SNARE complexes between vesicular VAMP and plasma membrane syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the plasma membrane. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion.  These findings reveal an insulin-dependent regulation of GLUT4 insertion into the plasma membrane involving complexin-2.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

scholarly journals Role of the Varicella-Zoster Virus gB Cytoplasmic Domain in gB Transport and Viral Egress

2002 ◽  
Vol 76 (2) ◽  
pp. 591-599 ◽  
Author(s):  
Thomas C. Heineman ◽  
Susan L. Hall
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Signal Sequence ◽  
Cytoplasmic Domain ◽  
Varicella Zoster ◽  
Golgi Transport ◽  
Viral Egress

ABSTRACT To study the function of the varicella-zoster virus (VZV) gB cytoplasmic domain during viral infection, we produced a VZV recombinant virus that expresses a truncated form of gB lacking the C-terminal 36 amino acids of its cytoplasmic domain (VZV gB-36). VZV gB-36 replicates in noncomplementing cells and grows at a rate similar to that of native VZV. However, cells infected with VZVgB-36 form extensive syncytia compared to the relatively small syncytia formed during native VZV infection. In addition, electron microscopy shows that very little virus is present on the surfaces of cells infected with VZV gB-36, while cells infected with native VZV exhibit abundant virions on the cell surface. The C-terminal 36 amino acids of the gB cytoplasmic domain have been shown in transfection-based experiments to contain both an endoplasmic reticulum-to-Golgi transport signal (the C-terminal 17 amino acids) and a consensus YXXφ (where Y is tyrosine, X is any amino acid, and φ is any bulky hydrophobic amino acid) signal sequence (YSRV) that mediates the internalization of gB from the plasma membrane. As predicted based on these data, gB-36 expressed during the infection of cultured cells is transported inefficiently to the Golgi. Despite lacking the YSRV signal sequence, gB-36 is internalized from the plasma membrane; however, in contrast to native gB, it fails to localize to the Golgi. Therefore, the C-terminal 36 amino acids of the VZV gB cytoplasmic domain are required for normal viral egress and for both the pre- and post-Golgi transport of gB.

scholarly journals Oligomerization of a membrane protein correlates with its retention in the Golgi complex

1993 ◽  
Vol 122 (6) ◽  
pp. 1185-1196 ◽  
Author(s):  
OA Weisz ◽  
AM Swift ◽  
CE Machamer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cytochalasin D ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Proteolytic Digestion ◽  
Golgi Transport ◽  
Large Oligomer ◽  
Membrane Spanning ◽  
Lumenal Domain

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

scholarly journals Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

2004 ◽  
Vol 165 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Matthew N.J. Seaman
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Multivesicular Body ◽  
Carboxypeptidase Y ◽  
Reporter Protein ◽  
Rapid Degradation ◽  
Endosomal Sorting ◽  
Lysosome Biogenesis ◽  
Golgi Transport ◽  
Efficient Retrieval

fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called “retromer” that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

scholarly journals A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

2014 ◽  
Vol 205 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Cortney C. Winkle ◽  
Leslie M. McClain ◽  
Juli G. Valtschanoff ◽  
Charles S. Park ◽  
Christopher Maglione ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cortical Neurons ◽  
Direct Interaction ◽  
Vesicle Fusion ◽  
Dependent Manner ◽  
Axon Branching ◽  
Spatial Regulation ◽  
Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

scholarly journals The Syntaxin 4 N Terminus Regulates Its Basolateral Targeting by Munc18c-dependent and -independent Mechanisms

2011 ◽  
Vol 286 (12) ◽  
pp. 10834-10846 ◽  
Author(s):  
Jacqueline Torres ◽  
Holly M. Funk ◽  
Mirjam M. P. Zegers ◽  
Martin B. A. ter Beest
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Basolateral Membrane ◽  
Vesicle Fusion ◽  
Secretory Proteins ◽  
Dependent Manner ◽  
Sm Proteins ◽  
N Terminus ◽  
Syntaxin 4 ◽  
Basolateral Targeting

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.

scholarly journals CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

2014 ◽  
Vol 25 (4) ◽  
pp. 508-521 ◽  
Author(s):  
Greg Kabachinski ◽  
Masaki Yamaga ◽  
D. Michelle Kielar-Grevstad ◽  
Stephen Bruinsma ◽  
Thomas F. J. Martin
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Total Internal Reflection ◽  
Vesicle Fusion ◽  
Live Cells ◽  
Membrane Domains ◽  
Direct View ◽  
Vesicle Exocytosis ◽  
Dependent Protein

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

scholarly journals Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

1994 ◽  
Vol 299 (2) ◽  
pp. 473-479 ◽  
Author(s):  
H Sengeløv ◽  
F Boulay ◽  
L Kjeldsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Beta Band ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

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