Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the
            trans
            ‐Golgi network but not the Golgi apparatus in Exo2‐treated cells

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0014 ◽

2013 ◽

Vol 24 (18) ◽

pp. 2907-2917 ◽

Cited By ~ 12

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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A hypothesis on the traffic of MG160, a medial Golgi sialoglycoprotein, from the trans-Golgi network to the Golgi cisternae

Journal of Cell Science ◽

10.1242/jcs.107.3.529 ◽

1994 ◽

Vol 107 (3) ◽

pp. 529-537 ◽

Cited By ~ 2

Author(s):

P.A. Johnston ◽

A. Stieber ◽

N.K. Gonatas

Keyword(s):

Sialic Acid ◽

Golgi Apparatus ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Brefeldin A ◽

Retrograde Transport ◽

Transport Pathway ◽

Trans Golgi Network ◽

Covalently Linked ◽

Golgi Network

We have reported that MG160, an intrinsic membrane sialoglycoprotein of the Golgi apparatus (GA), resides in the medial cisternae of the organelle (Gonatas et al. (1989) J. Biol. Chem. 264, 646–653). In order to resolve the question whether MG160 acquires sialic acid residues in the trans cisternae or trans-Golgi network (TGN) prior to its retrograde transport, we have examined the effects of brefeldin A (BFA) on the post-translational processing of MG160, and the distribution of internalized wheat germ agglutinin covalently linked with HRP (WGA-HRP), which labels the TGN (Gonatas et al. (1977) J. Cell Biol. 73, 1–13). In BFA-treated PC12 cells, MG160 acquires resistance to endo H, but fails to be sialylated. This effect occurs in parallel with the redistribution of MG160 into an ER compartment dispersed throughout the cytoplasm including the nuclear envelope, and the collapse of the WGA-HRP-labelled TGN into vesicles and tubules surrounding the centriole. These results suggest that MG160 is not sialylated in BFA-treated cells because it is sequestered from the sialyltransferase enzyme(s), presumably located in the TGN, and provide evidence supporting the hypothesis for a retrograde transport pathway that recycles resident GA proteins, including MG160, between the Golgi cisternae and the TGN. To examine further the above hypothesis we studied cells treated with BFA and then allowed to recover from the effect of the drug for various lengths of time. After 15 minutes of recovery, cisternae of the Golgi apparatus, typically found in the pericentriolar region, are labeled by both MG160 and WGA-HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

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Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

Journal of Cell Science ◽

10.1242/jcs.107.3.539 ◽

1994 ◽

Vol 107 (3) ◽

pp. 539-549 ◽

Cited By ~ 1

Author(s):

C.S. Velez-Granell ◽

A.E. Arias ◽

J.A. Torres-Ruiz ◽

M. Bendayan

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Secretory Pathway ◽

Secretory Granules ◽

Acinar Cells ◽

Pancreatic Tissue ◽

Secretory Proteins ◽

Trans Golgi Network ◽

Hsp70 Protein ◽

Golgi Network

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

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Regulation of Microtubule-dependent Recycling at the Trans-Golgi Network by Rab6A and Rab6A'

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0260 ◽

2005 ◽

Vol 16 (1) ◽

pp. 162-177 ◽

Cited By ~ 89

Author(s):

Joanne Young ◽

Tobias Stauber ◽

Elaine del Nery ◽

Isabelle Vernos ◽

Rainer Pepperkok ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Small Interfering Rna ◽

Small Gtpase ◽

Present Evidence ◽

Trans Golgi Network ◽

Endosomal Recycling ◽

Recycling Pathway ◽

Golgi Network ◽

Interfering Rna

The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A' has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A', binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A' GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins.

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μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

Journal of Cell Science ◽

10.1242/jcs.114.24.4469 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4469-4476 ◽

Cited By ~ 1

Author(s):

Christoph Meyer ◽

Eeva-Liisa Eskelinen ◽

Medigeshi Ramarao Guruprasad ◽

Kurt von Figura ◽

Peter Schu

Keyword(s):

Plasma Membrane ◽

Fold Increase ◽

Retrograde Transport ◽

Receptor Internalization ◽

Recycling Rate ◽

Coated Pits ◽

Trans Golgi Network ◽

Internalization Rate ◽

Clathrin Adaptor ◽

Golgi Network

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

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GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1687 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1687-1694 ◽

Cited By ~ 23

Author(s):

B Thorens ◽

N Gérard ◽

N Dériaz

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Beta Cells ◽

Intracellular Transport ◽

Brefeldin A ◽

Surface Expression ◽

Trans Golgi Network ◽

Intracellular Vesicles ◽

Insulinoma Cells ◽

Golgi Network

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

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