scholarly journals Participation of the Syntaxin 5/Ykt6/GS28/GS15 SNARE Complex in Transport from the Early/Recycling Endosome to the Trans-Golgi Network

2004 ◽  
Vol 15 (9) ◽  
pp. 4011-4022 ◽  
Author(s):  
Guihua Tai ◽  
Lei Lu ◽  
Tuan Lao Wang ◽  
Bor Luen Tang ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Snare Complex ◽  
Recycling Endosome ◽  
Trans Golgi Network ◽  
B Subunit ◽  
Protein Receptors ◽  
Transport Assay ◽  
Golgi Network ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.

scholarly journals Autoantigen Golgin-97, an Effector of Arl1 GTPase, Participates in Traffic from the Endosome to the Trans-Golgi Network

2004 ◽  
Vol 15 (10) ◽  
pp. 4426-4443 ◽  
Author(s):  
Lei Lu ◽  
Guihua Tai ◽  
Wanjin Hong
Keyword(s):  
Functional Study ◽  
Toxin B ◽  
Membrane Recruitment ◽  
Trans Golgi Network ◽  
Transport Assay ◽  
Retrograde Traffic ◽  
Golgi Network

The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite our recent understanding that Arl1 regulates the membrane recruitment of these Golgins. In this report, we describe our functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, we demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibited the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, was required in the in vitro transport assay. The kinetic characterization of inhibition by anti-Golgin-97 antibody in comparison with anti-Syntaxin 16 antibody established that Golgin-97 acts before Syntaxin 16 in endosome-to-TGN transport. Knock down of Golgin-97 or Arl1 by their respective small interference RNAs (siRNAs) also significantly inhibited the transport of STxB to the Golgi in vivo. In siRNA-treated cells with reduced levels of Arl1, internalized STxB was instead distributed peripherally. Microinjection of Golgin-97 antibody led to the fragmentation of Golgi apparatus and the arrested transport to the Golgi of internalized Cholera toxin B fragment. We suggest that Golgin-97 may function as a tethering molecule in endosome-to-TGN retrograde traffic.

scholarly journals A Novel Rab9 Effector Required for Endosome-to-TGN Transport

1997 ◽  
Vol 138 (2) ◽  
pp. 283-290 ◽  
Author(s):  
Elva Díaz ◽  
Frauke Schimmöller ◽  
Suzanne R. Pfeffer
Keyword(s):  
Active Form ◽  
Sucrose Density Gradient ◽  
Vesicle Docking ◽  
Trans Golgi Network ◽  
Yeast Two Hybrid System ◽  
Transport Assay ◽  
Transport Vesicle ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9–GTP with roughly fourfold preference to Rab9–GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (∼30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

scholarly journals Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 823-831 ◽  
Author(s):  
Y Goda ◽  
S R Pfeffer
Keyword(s):  
Early Stage ◽  
Gradient Centrifugation ◽  
Vesicular Transport ◽  
Free System ◽  
Transport Reaction ◽  
Trans Golgi Network ◽  
Transport Vesicles ◽  
Golgi Network ◽  
Gamma S

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

scholarly journals GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif

2003 ◽  
Vol 14 (3) ◽  
pp. 973-986 ◽  
Author(s):  
Annette M. Shewan ◽  
Ellen M. van Dam ◽  
Sally Martin ◽  
Tang Bor Luen ◽  
Wanjin Hong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Adipocyte Differentiation ◽  
Attachment Protein ◽  
Trans Golgi Network ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Endosomal System

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of thetrans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network

1999 ◽  
Vol 112 (6) ◽  
pp. 845-854 ◽  
Author(s):  
A.C. Valdez ◽  
J.P. Cabaniols ◽  
M.J. Brown ◽  
P.A. Roche
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Family Members ◽  
Snare Proteins ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Syntaxin 11 ◽  
Golgi Network

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro

Science ◽  
1990 ◽  
Vol 248 (4962) ◽  
pp. 1539-1541 ◽  
Author(s):  
R. Draper ◽  
Y Goda ◽  
F. Brodsky ◽  
Pfeffer
Keyword(s):  
Trans Golgi Network ◽  
Golgi Network

scholarly journals In vitro antibacterial activity and in vivo efficacy of sulbactam-durlobactam against pathogenic Burkholderia species

2020 ◽  
pp. AAC.01930-20
Author(s):  
Krisztina M. Papp-Wallace ◽  
Adam B. Shapiro ◽  
Scott A. Becka ◽  
Elise T. Zeiser ◽  
John J. LiPuma ◽  
...  
Keyword(s):  
Human Pathogens ◽  
Minimal Inhibitory Concentrations ◽  
Class A ◽  
In Vitro Antibacterial Activity ◽  
Lung Infections ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Inhibitor Combination

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, B. mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen β-lactamase, which confers resistance to β-lactams. The β-lactam-β-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. Herein, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured minimal inhibitory concentrations (MICs) of SUL-DUR against BCC and B. gladioli (N = 150), B. mallei (N = 30) and B. pseudomallei (N = 28); studied the kinetics of inhibition of the PenA1 β-lactamase from B. multivorans and the PenI β-lactamase from B. pseudomallei by durlobactam; tested for blaPenA1 induction by SUL-DUR; and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 μg/mL. Durlobactam potently inhibited PenA1 and PenI with k2/K values of 3.9 x 106 M−1s−1 and 2.6 x 103 M−1s−1 and Ki app of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest SUL-DUR may be useful as a treatment for Burkholderia infections.

scholarly journals Syntaxin 16’s Newly Deciphered Roles in Autophagy

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1655 ◽  
Author(s):  
Bor Luen Tang
Keyword(s):  
Membrane Trafficking ◽  
Functional Redundancy ◽  
Transport Processes ◽  
Snare Complex ◽  
Dual Roles ◽  
Trans Golgi Network ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Golgi Network

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16’s recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16’s newly found role in autophagy.

scholarly journals A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex

2013 ◽  
Vol 24 (18) ◽  
pp. 2907-2917 ◽  
Author(s):  
Kohei Arasaki ◽  
Daichi Takagi ◽  
Akiko Furuno ◽  
Miwa Sohda ◽  
Yoshio Misumi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Initial Contact ◽  
Complex Assembly ◽  
Trans Golgi Network ◽  
Cog Complex ◽  
Protein Receptor ◽  
Golgi Network

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor–mediated initial contact followed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum–localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to–trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

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