Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

10.3791/59111 ◽  
2019 ◽  
Author(s):  
Dominik P. Buser ◽  
Martin Spiess
Keyword(s):  
Cultured Cells ◽  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network

2005 ◽  
Vol 29 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Tina Wahle ◽  
Kai Prager ◽  
Nikolai Raffler ◽  
Christian Haass ◽  
Michael Famulok ◽  
...  
Keyword(s):  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

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.

Effect of monensin on plant Golgi: re-examination of the monensin-induced changes in cisternal architecture and functional activities of the Golgi apparatus of sycamore suspension-cultured cells

1993 ◽  
Vol 104 (3) ◽  
pp. 819-831 ◽  
Author(s):  
G.F. Zhang ◽  
A. Driouich ◽  
L.A. Staehelin
Keyword(s):  
High Pressure ◽  
Golgi Apparatus ◽  
Surface Area ◽  
Protein Secretion ◽  
Cultured Cells ◽  
Cellulose Synthesis ◽  
Trans Golgi Network ◽  
Suspension Cultured Cells ◽  
Atp Levels ◽  
Golgi Network

We have re-examined the effects of the ionophore monensin on the Golgi apparatus of sycamore maple suspension-cultured cells using a combination of high pressure freezing, immunocytochemical and biochemical techniques. Exposure of the cells to 10 microM monensin, which reduces protein secretion by approximately 90%, resulted first in the swelling of the trans-Golgi network, then of the trans-most trans-cisterna, the remaining trans-cisternae, and finally of the cis and medial cisternae. We postulate that these different rates of swelling reflect an underlying hierarchy of compartmental acidification with the trans-Golgi network being the most acidic compartment. Recovery occurred in the reverse sequence. Previous studies have suggested that the large swollen vesicles that accumulate in the cytoplasm of monensin-treated cells arise from the swelling and detachment of entire trans-cisternae. However, based on the many membrane blebbing configurations seen in association with the trans-Golgi network and the trans-Golgi cisternae of monensin-treated cells, and the fact that the surface area of the trans-Golgi cisternae is about five times greater than the surface area of the swollen vesicles, it appears that the swollen vesicles are produced by a budding mechanism. After 35–40 min of monensin treatment, cells with smaller, non-swollen, compact Golgi stacks began to appear and rapidly increased in number, contributing > 60% of the cell population after 60 min and > 80% after 100 min. In contrast, large numbers of swollen vesicles persisted in the cytoplasm of all cells for over 100 min. Since azide treatment of monensin-treated cells can prematurely induce the unswelling response and cellular ATP levels drop substantially after 45 min of monensin treatment, we propose that un-swelling of the Golgi stacks is due to a monensin-induced decline in ATP levels in the cells. Immunocytochemical labeling of the high pressure frozen cells with anti-xyloglucan antibodies demonstrated that the concentration of xyloglucan, a hemicellulose, in the swollen vesicles increased with time. This increase in vesicle contents may explain why these swollen vesicles do not contract in parallel with the Golgi stacks. In vivo labeling experiments with [3H]fucose, [3H]UDP-glucose and [3H]leucine demonstrated that monensin-treatment not only inhibited protein secretion, but also cellulose synthesis. Protein synthesis, on the other hand, was reduced only slightly during the first 30 min of treatment, but quite strongly between 30 and 60 min, consistent with the observed drop in ATP levels after > 40 min of exposure to monensin.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin

2001 ◽  
Vol 114 (2) ◽  
pp. 353-365 ◽  
Author(s):  
X. Zhao ◽  
T. Greener ◽  
H. Al-Hasani ◽  
S.W. Cushman ◽  
E. Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
J Domain ◽  
Almost All ◽  
Golgi Network ◽  
Assembly Proteins

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

scholarly journals Insulin-responsive amino peptidase follows the Glut4 pathway but is dispensable for the formation and translocation of insulin-responsive vesicles

2019 ◽  
Vol 30 (12) ◽  
pp. 1536-1543 ◽  
Author(s):  
Xiang Pan ◽  
Anatoli Meriin ◽  
Guanrong Huang ◽  
Konstantin V. Kandror
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Retrograde Transport ◽  
Skeletal Muscle Cells ◽  
Glucose Transporter 4 ◽  
Trans Golgi Network ◽  
Insulin Responsiveness ◽  
Golgi Network ◽  
Component Protein

In fat and skeletal muscle cells, insulin-responsive amino peptidase (IRAP) along with glucose transporter 4 (Glut4) and sortilin, represents a major component protein of the insulin-responsive vesicles (IRVs). Here, we show that IRAP, similar to Glut4 and sortilin, is retrieved from endosomes to the trans-Golgi network by retromer. Unlike Glut4, retrograde transport of IRAP does not require sortilin, as retromer can directly bind to the cytoplasmic tail of IRAP. Ablation of IRAP in 3T3-L1 adipocytes shifts the endosomal pool of Glut4 to more acidic endosomes, but does not affect IRV targeting, stability, and insulin responsiveness of Glut4.

scholarly journals Immunocytochemical Localization of the Menkes Copper Transport Protein (ATP7A) to the Trans-Golgi Network

1997 ◽  
Vol 6 (3) ◽  
pp. 409-416 ◽  
Author(s):  
Herman A. Dierick ◽  
Ayla N. Adam ◽  
June F. Escara-Wilke ◽  
Thomas W. Glover
Keyword(s):  
Golgi Complex ◽  
Cho Cells ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Western Blots ◽  
Fusion Construct ◽  
Copper Trafficking ◽  
Trans Golgi Network ◽  
Amino Terminal ◽  
Golgi Network

Abstract We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1–4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the ∼165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of α-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

scholarly journals Retrograde transport of γ-secretase from endosomes to the trans-Golgi network regulates Aβ42 production

2018 ◽  
Vol 147 (1) ◽  
pp. 110-123 ◽  
Author(s):  
Kunihiko Kanatsu ◽  
Yukiko Hori ◽  
Ihori Ebinuma ◽  
Yung Wen Chiu ◽  
Taisuke Tomita
Keyword(s):  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

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

1994 ◽  
Vol 107 (3) ◽  
pp. 529-537 ◽  
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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