scholarly journals Transition of Galactosyltransferase 1 from Trans-Golgi Cisterna to the Trans-Golgi Network Is Signal Mediated

2006 ◽  
Vol 17 (12) ◽  
pp. 5153-5162 ◽  
Author(s):  
Beat E. Schaub ◽  
Bea Berger ◽  
Eric G. Berger ◽  
Jack Rohrer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Time Lapse ◽  
Trans Golgi Network ◽  
Golgi Cisterna ◽  
Confocal Fluorescence ◽  
Rapid Accumulation ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Complex Glycan

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate β1,4-galactosyltransferase 1 (galT) and α2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT–siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

scholarly journals The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane.

1996 ◽  
Vol 132 (4) ◽  
pp. 565-576 ◽  
Author(s):  
J Rohrer ◽  
A Schweizer ◽  
D Russell ◽  
S Kornfeld
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Cytoplasmic Tail ◽  
Kinetic Studies ◽  
Type I ◽  
Trans Golgi Network ◽  
Intracellular Targeting ◽  
Sorting Motif ◽  
Golgi Network

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

scholarly journals Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

2004 ◽  
Vol 165 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Sebastien Carreno ◽  
Åsa E. Engqvist-Goldstein ◽  
Claire X. Zhang ◽  
Kent L. McDonald ◽  
David G. Drubin
Keyword(s):  
Time Lapse ◽  
Actin Dynamics ◽  
Coated Vesicle ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Trans Golgi Network ◽  
Lysosome Biogenesis ◽  
Diverse Species ◽  
Golgi Network ◽  
Golgi Organization

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

Multimeric connexin interactions prior to the trans-Golgi network

2001 ◽  
Vol 114 (22) ◽  
pp. 4013-4024
Author(s):  
Jayasri Das Sarma ◽  
Rita A. Meyer ◽  
Fushan Wang ◽  
Valsamma Abraham ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Dominant Negative ◽  
Alveolar Epithelial Cells ◽  
Trans Golgi Network ◽  
Alveolar Epithelial ◽  
C Terminus ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Nih 3T3 ◽  
Gap Junction Hemichannels

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial β-galactosidase fused to the C terminus of connexin43 (Cx43/β-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/β-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a subhexameric complex. Cx43/β-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/β-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/β-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines

2017 ◽  
Vol 96 (5) ◽  
pp. 432-439 ◽  
Author(s):  
Jeroen Declercq ◽  
Bruno Ramos-Molina ◽  
Ragna Sannerud ◽  
Bas Brouwers ◽  
Vincent P.E.G. Pruniau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Proprotein Convertase ◽  
Basic Amino Acids ◽  
Trans Golgi Network ◽  
Network Transport ◽  
Golgi Network

scholarly journals Soi3p/Rav1p Functions at the Early Endosome to Regulate Endocytic Trafficking to the Vacuole and Localization of Trans-Golgi Network Transmembrane Proteins

2004 ◽  
Vol 15 (7) ◽  
pp. 3196-3209 ◽  
Author(s):  
György Sipos ◽  
Jason H. Brickner ◽  
E.J. Brace ◽  
Linyi Chen ◽  
Alain Rambourg ◽  
...  
Keyword(s):  
Vacuolar Atpase ◽  
Early Endosome ◽  
Trans Golgi Network ◽  
Peripheral Protein ◽  
Early Endosomes ◽  
Localization Signal ◽  
Prevacuolar Compartment ◽  
Gradient Fractionation ◽  
Vacuolar Protein ◽  
Golgi Network

SOI3 was identified by a mutation, soi3-1, that suppressed a mutant trans-Golgi network (TGN) localization signal in the Kex2p cytosolic tail. SOI3, identical to RAV1, encodes a protein important for regulated assembly of vacuolar ATPase. Here, we show that Soi3/Rav1p is required for transport between the early endosome and the late endosome/prevacuolar compartment (PVC). By electron microscopy, soi3-1 mutants massively accumulated structures that resembled early endosomes. soi3Δ mutants exhibited a kinetic delay in transfer of the endocytic tracer dye FM4-64, from the 14°C endocytic intermediate to the vacuole. The soi3Δ mutation delayed vacuolar degradation but not internalization of the a-factor receptor Ste3p. By density gradient fractionation, Soi3/Rav1p associated as a peripheral protein with membranes of a density characteristic of early endosomes. The soi3 null mutation markedly reduced the rate of Kex2p transport from the TGN to the PVC but had no effect on vacuolar protein sorting or cycling of Vps10p. These results suggest that assembly of vacuolar ATPase at the early endosome is required for transport of both Ste3p and Kex2p from the early endosome to the PVC and support a model in which cycling through the early endosome is part of the normal itinerary of Kex2p and other TGN-resident proteins.

Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization

2000 ◽  
Vol 352 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Jan-Willem H. P. VAN DE LOO ◽  
Meike TEUCHERT ◽  
Ilse PAULI ◽  
Evelyn PLETS ◽  
Wim J. M.VAN DE VEN ◽  
...  
Keyword(s):  
Half Life ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Cysteine Residue ◽  
Type I ◽  
Proprotein Convertase ◽  
Trans Golgi Network ◽  
Golgi Network

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t1/2 ≈ 50min) than that of the LPC polypeptide backbone (t1/2 ≈ 3h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Protein phosphatase 2A binds to the cytoplasmic tail of carboxypeptidase D and regulates post-trans-Golgi network trafficking

2001 ◽  
Vol 114 (2) ◽  
pp. 311-322
Author(s):  
O. Varlamov ◽  
E. Kalinina ◽  
F.Y. Che ◽  
L.D. Fricker
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cytoplasmic Tail ◽  
Trans Golgi Network ◽  
Phosphatase 2A ◽  
Carboxypeptidase D ◽  
Golgi Network

Carboxypeptidase D (CPD) is a transmembrane protein that processes proteins in the trans-Golgi network (TGN). A 20-residue region within the cytoplasmic tail of CPD binds protein phosphatase 2A (PP2A). PP2A also binds to the cytoplasmic tails of other secretory pathway proteins: peptidylglycine-(amino)-amidating mono-oxygenase, the cation-independent mannose-6-phosphate receptor and TGN38. The CPD tail is phosphorylated on Thr residues in the AtT-20 cell line. The CPD tail can also be phosphorylated by purified protein kinase A, protein kinase C and casein kinase II. Both the in vitro and the in vivo phosphorylated CPD tail can be dephosphorylated by purified PP2A. The binding of CPD tail peptide to PP2A does not influence phosphatase activity. The rate of transport of CPD from the TGN to the cell surface of AtT-20 cells is decreased 45% by okadaic acid, a PP2A inhibitor. Microinjection of the CPD tail into AtT-20 cells inhibits the transition of CPD from endosomal compartments to the TGN. However, okadaic acid does not affect the rate of budding of CPD from the TGN into nascent vesicles or the rate of uptake from the cell surface into endosomal compartments. These results are consistent with the model that PP2A is involved in the trafficking of proteins between a TGN recycling loop and a cell-surface recycling loop, but is not involved in the individual recycling loops.

scholarly journals Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells.

1995 ◽  
Vol 128 (3) ◽  
pp. 321-332 ◽  
Author(s):  
S Höning ◽  
W Hunziker
Keyword(s):  
Mdck Cells ◽  
Transmembrane Protein ◽  
Cytoplasmic Tail ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Lysosomal Sorting ◽  
Lysosomal System ◽  
Golgi Network ◽  
Basolateral Targeting ◽  
Cytoplasmic Determinants

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.

scholarly journals Visualization of secretory cargo transport within the Golgi apparatus

2019 ◽  
Vol 218 (5) ◽  
pp. 1602-1618 ◽  
Author(s):  
Kazuo Kurokawa ◽  
Hiroko Osakada ◽  
Tomoko Kojidani ◽  
Miho Waga ◽  
Yasuyuki Suda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Golgi Cisterna ◽  
Cargo Transport ◽  
Golgi Network ◽  
Interesting Behavior

To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone.

scholarly journals The Cytoplasmic Tail of α1,2-Fucosyltransferase Contains a Sequence for Golgi Localization

2001 ◽  
Vol 276 (15) ◽  
pp. 12012-12018 ◽  
Author(s):  
Julie Milland ◽  
Simon G. Taylor ◽  
Hayley C. Dodson ◽  
Ian F. C. McKenzie ◽  
Mauro S. Sandrin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cytoplasmic Tail ◽  
Single Amino Acid ◽  
Predominant Role ◽  
Golgi Localization ◽  
Sequential Addition ◽  
Golgi Network ◽  
Flanking Regions

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of α1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the α1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of α1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser5. Mutagenesis of Ser5to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of α1,2-fucosyltransferase.

Sign in / Sign up

Export Citation Format

Share Document