scholarly journals Identification, molecular characterization and immunolocalization of an isoform of the trans-Golgi-network (TGN)-specific integral membrane protein TGN38

1992 ◽  
Vol 283 (2) ◽  
pp. 313-316 ◽  
Author(s):  
B Reaves ◽  
A Wilde ◽  
G Banting
Keyword(s):  
Membrane Protein ◽  
Molecular Characterization ◽  
Integral Membrane Protein ◽  
Cdna Clones ◽  
Protein Coding ◽  
Trans Golgi Network ◽  
Coding Regions ◽  
Cytoplasmic Tails ◽  
Localization Signal ◽  
Golgi Network

TGN38 is an integral membrane protein previously shown to be predominantly localized to the trans-Golgi network (TGN) of cells by virtue of a signal contained within its cytoplasmic ‘tail’ [Luzio, Brake, Banting, Howell, Braghetta & Stanley (1990) Biochem. J. 270, 97-102]. We now (i) describe the isolation of cDNA clones encoding an isoform of TGN38, (ii) present the sequence of that isoform and (iii) describe the production and use of antibodies which specifically recognize the new isoform. This isoform, designated TGN41, is also predominantly localized to the TGN. The only sequence differences between the protein coding regions of cDNA clones encoding TGN38 and those encoding TGN41 occur within the region specifying the cytoplasmic tails of the two proteins. The TGN localization signal is shown to be within the sequence common to both proteins.

scholarly journals Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38)

1990 ◽  
Vol 270 (1) ◽  
pp. 97-102 ◽  
Author(s):  
J P Luzio ◽  
B Brake ◽  
G Banting ◽  
K E Howell ◽  
P Braghetta ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Trans Golgi Network ◽  
Sequencing And Expression ◽  
Novel Strategy ◽  
Golgi Network

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

scholarly journals Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38.

1992 ◽  
Vol 116 (1) ◽  
pp. 85-94 ◽  
Author(s):  
B Reaves ◽  
G Banting
Keyword(s):  
Membrane Protein ◽  
Morphological Changes ◽  
Brefeldin A ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Atp Depletion ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Organizing Center ◽  
Golgi Network

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.

scholarly journals MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

1999 ◽  
Vol 10 (10) ◽  
pp. 3435-3447 ◽  
Author(s):  
Rosa Puertollano ◽  
Miguel A. Alonso
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Ectopic Expression ◽  
Integral Membrane Protein ◽  
Membrane Microdomains ◽  
Surface Binding ◽  
Flag Epitope ◽  
Trans Golgi Network ◽  
Endosomal Acidification ◽  
Golgi Network

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane–mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH4Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that ∼30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

scholarly journals A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network

1993 ◽  
Vol 122 (4) ◽  
pp. 775-788 ◽  
Author(s):  
SM Jones ◽  
JR Crosby ◽  
J Salamero ◽  
KE Howell
Keyword(s):  
Binding Proteins ◽  
Integral Membrane Protein ◽  
Preliminary Evidence ◽  
Velocity Sedimentation ◽  
Free System ◽  
Gtp Binding Proteins ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Transport Vesicles ◽  
Golgi Network

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.

scholarly journals Influenza B Virus BM2 Protein Is Transported through the trans-Golgi Network as an Integral Membrane Protein

2003 ◽  
Vol 77 (19) ◽  
pp. 10630-10637 ◽  
Author(s):  
Shinji Watanabe ◽  
Masaki Imai ◽  
Yoshiro Ohara ◽  
Takato Odagiri
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Matrix Protein ◽  
Integral Membrane Protein ◽  
Influenza B Virus ◽  
Influenza B ◽  
Hydrophobic Domain ◽  
Cytoplasmic Transport ◽  
B Virus ◽  
Golgi Network

ABSTRACT A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.

scholarly journals VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.

1992 ◽  
Vol 118 (5) ◽  
pp. 1003-1014 ◽  
Author(s):  
T V Kurzchalia ◽  
P Dupree ◽  
R G Parton ◽  
R Kellner ◽  
H Virta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Cellular Membrane ◽  
Integral Membrane Proteins ◽  
Membrane Domains ◽  
Transport Vesicles ◽  
Vesicular Carriers ◽  
Molecular Machinery ◽  
Golgi Network

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

scholarly journals A Modulatory Role for Clathrin Light Chain Phosphorylation in Golgi Membrane Protein Localization during Vegetative Growth and during the Mating Response ofSaccharomyces cerevisiae

1999 ◽  
Vol 10 (3) ◽  
pp. 713-726 ◽  
Author(s):  
Diana S. Chu ◽  
Babak Pishvaee ◽  
Gregory S. Payne
Keyword(s):  
Signal Transduction ◽  
Membrane Protein ◽  
Light Chain ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Response Signal ◽  
Mating Pheromone ◽  
Mating Response ◽  
Trans Golgi Network ◽  
Golgi Network

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the α-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.

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